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Dalla Pozza M, Abdullrahman A, Cardin CJ, Gasser G, Hall JP. Three's a crowd - stabilisation, structure, and applications of DNA triplexes. Chem Sci 2022; 13:10193-10215. [PMID: 36277639 PMCID: PMC9473520 DOI: 10.1039/d2sc01793h] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Accepted: 08/02/2022] [Indexed: 12/16/2022] Open
Abstract
DNA is a strikingly flexible molecule and can form a variety of secondary structures, including the triple helix, which is the subject of this review. The DNA triplex may be formed naturally, during homologous recombination, or can be formed by the introduction of a synthetic triplex forming oligonucleotide (TFO) to a DNA duplex. As the TFO will bind to the duplex with sequence specificity, there is significant interest in developing TFOs with potential therapeutic applications, including using TFOs as a delivery mechanism for compounds able to modify or damage DNA. However, to combine triplexes with functionalised compounds, a full understanding of triplex structure and chemical modification strategies, which may increase triplex stability or in vivo degradation, is essential - these areas will be discussed in this review. Ruthenium polypyridyl complexes, which are able to photooxidise DNA and act as luminescent DNA probes, may serve as a suitable photophysical payload for a TFO system and the developments in this area in the context of DNA triplexes will also be reviewed.
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Affiliation(s)
- Maria Dalla Pozza
- Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology F-75005 Paris France www.gassergroup.com
| | - Ahmad Abdullrahman
- Department of Pharmacy, Chemistry and Pharmacy Building, University of Reading Whiteknights Campus Reading Berkshire RG6 6AD UK
| | - Christine J Cardin
- Department of Chemistry, University of Reading Whiteknights Reading RG6 6AD UK
| | - Gilles Gasser
- Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology F-75005 Paris France www.gassergroup.com
| | - James P Hall
- Department of Pharmacy, Chemistry and Pharmacy Building, University of Reading Whiteknights Campus Reading Berkshire RG6 6AD UK
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Beyond G-Quadruplexes-The Effect of Junction with Additional Structural Motifs on Aptamers Properties. Int J Mol Sci 2021; 22:ijms22189948. [PMID: 34576112 PMCID: PMC8466185 DOI: 10.3390/ijms22189948] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 09/08/2021] [Accepted: 09/09/2021] [Indexed: 12/02/2022] Open
Abstract
G-quadruplexes constitute an important type of nucleic acid structure, which can be found in living cells and applied by cell machinery as pivotal regulatory elements. Importantly, robust development of SELEX technology and modern, nucleic acid-based therapeutic strategies targeted towards various molecules have also revealed a large group of potent aptamers whose structures are grounded in G-quadruplexes. In this review, we analyze further extension of tetraplexes by additional structural elements and investigate whether G-quadruplex junctions with duplex, hairpin, triplex, or second G-quadruplex motifs are favorable for aptamers stability and biological activity. Furthermore, we indicate the specific and pivotal role of the G-quadruplex domain and the additional structural elements in interactions with target molecules. Finally, we consider the potency of G-quadruplex junctions in future applications and indicate the emerging research area that is still waiting for development to obtain highly specific and effective nucleic acid-based molecular tools.
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3
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Riccardi C, Napolitano E, Platella C, Musumeci D, Montesarchio D. G-quadruplex-based aptamers targeting human thrombin: Discovery, chemical modifications and antithrombotic effects. Pharmacol Ther 2020; 217:107649. [PMID: 32777331 DOI: 10.1016/j.pharmthera.2020.107649] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2020] [Accepted: 07/29/2020] [Indexed: 02/07/2023]
Abstract
First studies on thrombin-inhibiting DNA aptamers were reported in 1992, and since then a large number of anticoagulant aptamers has been discovered. TBA - also named HD1, a 15-mer G-quadruplex (G4)-forming oligonucleotide - is the best characterized thrombin binding aptamer, able to specifically recognize the protein exosite I, thus inhibiting the conversion of soluble fibrinogen into insoluble fibrin strands. Unmodified nucleic acid-based aptamers, in general, and TBA in particular, exhibit limited pharmacokinetic properties and are rapidly degraded in vivo by nucleases. In order to improve the biological performance of aptamers, a widely investigated strategy is the introduction of chemical modifications in their backbone at the level of the nucleobases, sugar moieties or phosphodiester linkages. Besides TBA, also other thrombin binding aptamers, able to adopt a well-defined G4 structure, e.g. mixed duplex/quadruplex sequences, as well as homo- and hetero-bivalent constructs, have been identified and optimized. Considering the growing need of new efficient anticoagulant agents associated with the strong therapeutic potential of these thrombin inhibitors, the research on thrombin binding aptamers is still a very hot and intriguing field. Herein, we comprehensively described the state-of-the-art knowledge on the DNA-based aptamers targeting thrombin, especially focusing on the optimized analogues obtained by chemically modifying the oligonucleotide backbone, and their biological performances in therapeutic applications.
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Affiliation(s)
- Claudia Riccardi
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy; Department of Advanced Medical and Surgical Sciences, 2(nd) Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini, 5, I-80131 Naples, Italy.
| | - Ettore Napolitano
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy.
| | - Chiara Platella
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy.
| | - Domenica Musumeci
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy; Institute of Biostructures and Bioimages, CNR, via Mezzocannone 16, I-80134 Naples, Italy.
| | - Daniela Montesarchio
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy.
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Mao-Draayer Y, Thiel S, Mills EA, Chitnis T, Fabian M, Katz Sand I, Leite MI, Jarius S, Hellwig K. Neuromyelitis optica spectrum disorders and pregnancy: therapeutic considerations. Nat Rev Neurol 2020; 16:154-170. [PMID: 32080393 DOI: 10.1038/s41582-020-0313-y] [Citation(s) in RCA: 59] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/15/2020] [Indexed: 12/18/2022]
Abstract
Neuromyelitis optica spectrum disorders (NMOSD) are a type of neurological autoimmune disease characterized by attacks of CNS inflammation that are often severe and predominantly affect the spinal cord and optic nerve. The majority of individuals with NMOSD are women, many of whom are of childbearing age. Although NMOSD are rare, several small retrospective studies and case reports have indicated that pregnancy can worsen disease activity and might contribute to disease onset. NMOSD disease activity seems to negatively affect pregnancy outcomes. Moreover, some of the current NMOSD treatments are known to pose risks to the developing fetus and only limited safety data are available for others. Here, we review published studies regarding the relationship between pregnancy outcomes and NMOSD disease activity. We also assess the risks associated with using disease-modifying therapies for NMOSD during the course of pregnancy and breastfeeding. On the basis of the available evidence, we offer recommendations regarding the use of these therapies in the course of pregnancy planning in individuals with NMOSD.
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Affiliation(s)
- Yang Mao-Draayer
- Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.,Graduate Program in Immunology, Program in Biomedical Sciences, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Sandra Thiel
- Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany
| | - Elizabeth A Mills
- Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Tanuja Chitnis
- Department of Neurology, Brigham and Women's Hospital and Massachusetts General Hospital, Boston, MA, USA
| | - Michelle Fabian
- Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Ilana Katz Sand
- Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - M Isabel Leite
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
| | - Sven Jarius
- Molecular Neuroimmunology Group, Department of Neurology, University of Heidelberg, Heidelberg, Germany
| | - Kerstin Hellwig
- Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.
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5
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Sun Z, Wang X, Zhang JZH, He Q. Sulfur-substitution-induced base flipping in the DNA duplex. Phys Chem Chem Phys 2019; 21:14923-14940. [PMID: 31233058 DOI: 10.1039/c9cp01989h] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Base flipping is widely observed in a number of important biological processes. The genetic codes deposited inside the DNA duplex become accessible to external agents upon base flipping. The sulfur substitution of guanine leads to thioguanine, which alters the thermodynamic stability of the GC base pairs and the GT mismatches. Experimental studies conclude that the sulfur substitution decreases the lifetime of the GC base pair. In this work, under three AMBER force fields for nucleotide systems, we firstly performed equilibrium and nonequilibrium free energy simulations to investigate the variation of the thermodynamic profiles in base flipping upon sulfur substitution. It is found that the bsc0 modification, the bsc1 modification and the OL15 modification of AMBER force fields are able to qualitatively describe the sulfur-substitution dependent behavior of the thermodynamics. However, only the two last-generation AMBER force fields are able to provide quantitatively correct predictions. The second computational study on the sulfur substitutions focused on the relative stability of the S6G-C base pair and the S6G-T mismatch. Two conflicting experimental observations were reported by the same authors. One suggested that the S6G-C base pair was more stable, while the other concludes that the S6G-T mismatch was more stable. We answered this question by constructing the free energy profiles along the base flipping pathway computationally.
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Affiliation(s)
- Zhaoxi Sun
- State Key Laboratory of Precision Spectroscopy, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China and Computational Biomedicine (IAS-5/INM-9), Forschungszentrum Jülich, Jülich 52425, Germany.
| | - Xiaohui Wang
- State Key Laboratory of Precision Spectroscopy, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China and Institute of Computational Science, Università della Svizzera italiana (USI), Via Giuseppe Buffi 13, CH-6900, Lugano, Ticino, Switzerland
| | - John Z H Zhang
- State Key Laboratory of Precision Spectroscopy, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China and NYU-ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai 200062, China and Department of Chemistry, New York University, NY, NY 10003, USA
| | - Qiaole He
- Forschungszentrum Jülich GmbH, IBG-1: Biotechnology, Wilhelm-Johnen-Str. 1, 52425 Jülich, Germany. and State Key Laboratory of Bioreactor Engineering, R&D Center of Separation and Extraction Technology in Fermentation Industry, East China University of Science and Technology, Shanghai 200237, China
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6
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Analytical Pitfalls of Therapeutic Drug Monitoring of Thiopurines in Patients With Inflammatory Bowel Disease. Ther Drug Monit 2018; 39:584-588. [PMID: 29040228 PMCID: PMC5690305 DOI: 10.1097/ftd.0000000000000455] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The use of thiopurines in the treatment of inflammatory bowel disease (IBD) can be optimized by the application of therapeutic drug monitoring. In this procedure, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) metabolites are monitored and related to therapeutic response and adverse events, respectively. Therapeutic drug monitoring of thiopurines, however, is hampered by several analytical limitations resulting in an impaired translation of metabolite levels to clinical outcome in IBD. Thiopurine metabolism is cell specific and requires nucleated cells and particular enzymes for 6-TGN formation. In the current therapeutic drug monitoring, metabolite levels are assessed in erythrocytes, whereas leukocytes are considered the main target cells of these drugs. Furthermore, currently used methods do not distinguish between active nucleotides and their unwanted residual products. Last, there is a lack of a standardized laboratorial procedure for metabolite assessment regarding the substantial instability of erythrocyte 6-TGN. To improve thiopurine therapy in patients with IBD, it is necessary to understand these limitations and recognize the general misconceptions in this procedure.
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7
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Lunn SML, Hribesh S, Whitfield CJ, Hall MJ, Houlton A, Bronowska AK, Tuite EM, Pike AR. Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd 2+ Chemical Stimulus. Chembiochem 2018; 19:1115-1118. [PMID: 29575493 DOI: 10.1002/cbic.201800100] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Indexed: 11/08/2022]
Abstract
The on-column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio-directed Cd2+ coordination; a route for healing DNA damage. Short oligomers are G-specifically thiolated through a modified on-column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd2+ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3'-A6 tG4 T6 -5', suggests a (tG)8 Cd2 central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal-induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd2+ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer.
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Affiliation(s)
- Samantha M L Lunn
- School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK
| | - Samira Hribesh
- Department of Chemistry, Faculty of Arts and Sciences, Al-Mergeb University, Al-Khums, Libya
| | | | - Michael J Hall
- School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK
| | - Andrew Houlton
- School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK
| | | | - Eimer M Tuite
- School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK
| | - Andrew R Pike
- School of Chemistry, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK
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Guillotin V, Galli G, Viallard JF. [Usefulness of thiopurine methyltransferase polymorphism study and metabolites measurement for patients treated by azathioprine]. Rev Med Interne 2018; 39:421-426. [PMID: 29370945 DOI: 10.1016/j.revmed.2017.12.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2017] [Revised: 12/18/2017] [Accepted: 12/28/2017] [Indexed: 11/18/2022]
Abstract
Azathioprine is widely used in internal medicine and frequently implicated in occurrence of adverse events. Among these adverse events the bone marrow suppression, a dose-related one, is the most serious because of is potential morbidity and mortality. Severe myelosuppression, associated with abnormal AZA metabolism, is linked to the thiopurine methyltransferase (TPMT) genetic polymorphism that results in a high variability of its activity with 89% of patients with a normal activity, 11% with an intermediate activity, and 0.3% with very low activity leading to a very high risk of bonne marrow suppression. TPMT status can be assessed prior to AZA treatment by measuring enzyme activity or genotyping techniques to identify patients for which the standard dose is not advisable. Furthermore, azathioprine metabolites monitoring is helpful for the follow up of patients, especially in therapeutic failure, to distinguish non-compliant patients from under-dosed, "shunters" or resistant patients.
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Affiliation(s)
- V Guillotin
- Laboratoire d'immunologie de l'hôpital Pellegrin, place Amélié-Raba-Léon, 33000 Bordeaux, France.
| | - G Galli
- Service de médecine interne et immunologie clinique, Hôpital Saint-André, rue Jean-Burguet, 33000 Bordeaux, France
| | - J-F Viallard
- Service de médecine interne, hôpital du Haut-Lévêque, avenue Magellan, 33604 Pessac, France
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9
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Wyatt MD, Reilly NM, Patel S, Rajesh P, Schools GP, Smiraldo PG, Pittman DL. Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2018; 59:38-48. [PMID: 28945288 PMCID: PMC5771848 DOI: 10.1002/em.22138] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Revised: 06/09/2017] [Accepted: 08/09/2017] [Indexed: 06/07/2023]
Abstract
Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Michael D. Wyatt
- Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia SC 29208
| | - Nicole M. Reilly
- Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia SC 29208
| | - Shikha Patel
- Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia SC 29208
| | - Preeti Rajesh
- Department of Biotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib, Punjab, India-140406
| | - Gary P. Schools
- Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia SC 29208
| | | | - Douglas L. Pittman
- Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia SC 29208
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Dvořáková Z, Vorlíčková M, Renčiuk D. Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage. Biochim Biophys Acta Gen Subj 2017; 1861:2750-2757. [DOI: 10.1016/j.bbagen.2017.07.019] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Revised: 06/16/2017] [Accepted: 07/24/2017] [Indexed: 11/26/2022]
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Sagi J. In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids? J Nucleic Acids 2017; 2017:1641845. [PMID: 29181193 PMCID: PMC5664352 DOI: 10.1155/2017/1641845] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2017] [Accepted: 08/15/2017] [Indexed: 01/03/2023] Open
Abstract
Synthetic analogs of natural nucleotides have long been utilized for structural studies of canonical and noncanonical nucleic acids, including the extensively investigated polymorphic G-quadruplexes (GQs). Dependence on the sequence and nucleotide modifications of the folding landscape of GQs has been reviewed by several recent studies. Here, an overview is compiled on the thermodynamic stability of the modified GQ folds and on how the stereochemical preferences of more than 70 synthetic and natural derivatives of nucleotides substituting for natural ones determine the stability as well as the conformation. Groups of nucleotide analogs only stabilize or only destabilize the GQ, while the majority of analogs alter the GQ stability in both ways. This depends on the preferred syn or anti N-glycosidic linkage of the modified building blocks, the position of substitution, and the folding architecture of the native GQ. Natural base lesions and epigenetic modifications of GQs explored so far also stabilize or destabilize the GQ assemblies. Learning the effect of synthetic nucleotide analogs on the stability of GQs can assist in engineering a required stable GQ topology, and exploring the in vitro action of the single and clustered natural base damage on GQ architectures may provide indications for the cellular events.
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Affiliation(s)
- Janos Sagi
- Rimstone Laboratory, RLI, Carlsbad, CA 92010, USA
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12
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Lord JD, Shows DM. Thiopurine use associated with reduced B and natural killer cells in inflammatory bowel disease. World J Gastroenterol 2017; 23:3240-3251. [PMID: 28566883 PMCID: PMC5434429 DOI: 10.3748/wjg.v23.i18.3240] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Revised: 01/27/2017] [Accepted: 03/15/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To identify which blood and mucosal lymphocyte populations are specifically depleted by thiopurine use in vivo.
METHODS The thiopurines azathioprine and 6-mercaptopurine have been a mainstay of inflammatory bowel disease (IBD) therapy for decades, but their mechanism of action in vivo remains obscure. Although thiopurines are lymphotoxic at high doses, and have been reported to cause T cell apoptosis in vitro, their ability to control IBD at lower doses suggests that they may selectively deplete particular lymphocyte populations. Blood cells from 19 IBD patients on a thiopurine, 19 IBD patients not on a thiopurine, and 38 matched healthy control subjects were analyzed by multiple multi-color flow cytometry panels to quantify the immune cell subsets contained therein, both as a percent of cells, and as an absolute cell count. Similar analyses were performed on colon biopsies from 17 IBD patients on a thiopurine, 17 IBD patients not on a thiopurine, and 49 healthy screening colonoscopy recipients.
RESULTS Complete blood counts revealed lower lymphocyte, but not monocyte or granulocyte, counts in IBD patients who were taking thiopurines at the time of sampling. This reduction was restricted to CD3-negative lymphocytes, wherein both natural killer (NK) and B cells were significantly reduced among thiopurine recipients. Among CD19+ B cells, the transitional B cells were particularly depleted, being nearly absent in both blood and colon biopsies of thiopurine recipients. No differences were associated with thiopurine use in CD8+ T cells, mucosa-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, gamma/delta T cells, Th1, Th17, regulatory T cells (Tregs) or naïve CD4+ T cells. However, patients with IBD had significantly more circulating FOXP3+, Helios+ Tregs and fewer iNKT and MAIT cells than healthy controls.
CONCLUSION Thiopurine use is associated with reduced B and NK cell, but not T cell, subpopulations in the blood of IBD patients.
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13
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Liao JY, Anosova I, Bala S, Van Horn WD, Chaput JC. A parallel stranded G-quadruplex composed of threose nucleic acid (TNA). Biopolymers 2017; 107. [PMID: 27718227 DOI: 10.1002/bip.22999] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2016] [Revised: 09/26/2016] [Accepted: 10/05/2016] [Indexed: 11/08/2022]
Abstract
G-rich sequences can adopt four-stranded helical structures, called G-quadruplexes, that self-assemble around monovalent cations like sodium (Na+ ) and potassium (K+ ). Whether similar structures can be formed from xeno-nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α-l-threofuranosyl nucleic acid) to adopt a four-stranded helical structure based on a planar G-quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution-state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self-assemble into stable G-quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G-quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self-assembling into stable G-quadruplex structures.
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Affiliation(s)
- Jen-Yu Liao
- Department of Pharmaceutical Sciences, University of California, Irvine, CA, 92697
| | - Irina Anosova
- School of Molecular Sciences and the Biodesign Institute, Arizona State University. Tempe, AZ, 85287
| | - Saikat Bala
- Department of Pharmaceutical Sciences, University of California, Irvine, CA, 92697
| | - Wade D Van Horn
- School of Molecular Sciences and the Biodesign Institute, Arizona State University. Tempe, AZ, 85287
| | - John C Chaput
- Department of Pharmaceutical Sciences, University of California, Irvine, CA, 92697
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14
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Kosbar TR, Sofan MA, Abou-Zeid L, Pedersen EB. Thermal stability of G-rich anti-parallel DNA triplexes upon insertion of LNA and α-L-LNA. Org Biomol Chem 2016; 13:5115-21. [PMID: 25833006 DOI: 10.1039/c5ob00535c] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
G-rich anti-parallel DNA triplexes were modified with LNA or α-L-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-L-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-L-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-L-LNA the ΔTm increased. Moreover, increasing the number of α-L-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-L-LNA in anti-parallel triplexes is preferable for triplex stability.
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Affiliation(s)
- Tamer R Kosbar
- Nucleic Acid Center, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
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Cheong VV, Heddi B, Lech CJ, Phan AT. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad. Nucleic Acids Res 2015; 43:10506-14. [PMID: 26400177 PMCID: PMC4666386 DOI: 10.1093/nar/gkv826] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Accepted: 07/28/2015] [Indexed: 01/31/2023] Open
Abstract
G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions.
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Affiliation(s)
- Vee Vee Cheong
- School of Physical and Mathematical Sciences, Nanyang Technological University, 637371 Singapore
| | - Brahim Heddi
- School of Physical and Mathematical Sciences, Nanyang Technological University, 637371 Singapore
| | - Christopher Jacques Lech
- School of Physical and Mathematical Sciences, Nanyang Technological University, 637371 Singapore
| | - Anh Tuân Phan
- School of Physical and Mathematical Sciences, Nanyang Technological University, 637371 Singapore
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Nowakowska M, Kowalska J, Martin F, d'Orchymont A, Zuberek J, Lukaszewicz M, Darzynkiewicz E, Jemielity J. Cap analogs containing 6-thioguanosine--reagents for the synthesis of mRNAs selectively photo-crosslinkable with cap-binding biomolecules. Org Biomol Chem 2015; 12:4841-7. [PMID: 24763507 DOI: 10.1039/c4ob00059e] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Numerous biomolecules recognize the 7-methylguanosine cap structure present at the 5' ends of eukaryotic mRNAs. Photo-crosslinking is a valuable technique to study these interactions. We report three anti-reverse cap analogs containing a photo-activable nucleoside, 6-thioguanosine ((6S)G), that enable the synthesis of capped RNAs with (6S)G positioned exclusively as the first transcribed nucleotide. The effect of the 6-thioguanosine moiety on binding to the translation factor eIF4E and the efficiency of mRNA translation was determined. The utility of mRNAs with a (6S)G-modified cap in crosslinking experiments is shown by mapping the histone H4 cap-binding pocket.
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Affiliation(s)
- Monika Nowakowska
- Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland
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Miller MC, Ohrenberg CJ, Kuttan A, Trent JO. Separation of Quadruplex Polymorphism in DNA Sequences by Reversed-Phase Chromatography. CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY 2015; 61:17.7.1-17.7.18. [PMID: 26344226 PMCID: PMC4561857 DOI: 10.1002/0471142700.nc1707s61] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
This unit describes a method for the separation of a mixture of quadruplex conformations formed from the same parent sequence via reversed-phase chromatography (RPC). Polymorphism is inherent to quadruplex formation and even relatively simple quadruplex-forming sequences can fold into a cornucopia of possible conformations and topologies. Isolation of a specific conformation for study can be problematic. This is especially true for conformations of the human telomere sequence d(GGG(TTAGGG)3). High performance liquid chromatography (HPLC), especially reversed-phase chromatography, has been a mainstay of nucleic acid research and purification for many decades. We have successfully applied this method to the problem of separating individual quadruplex species in the ensemble from the same parent sequence.
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Affiliation(s)
- M. Clarke Miller
- Department of Chemistry and Biochemistry, University of North Georgia, 3820 Mundy Mill Road, Oakwood, Georgia 30566
- James Graham Brown Cancer Center, Clinical Translational Research Building, University of Louisville, 505 South Hancock Street, Louisville, KY 40202
| | - Carl J. Ohrenberg
- Department of Chemistry and Biochemistry, University of North Georgia, 3820 Mundy Mill Road, Oakwood, Georgia 30566
| | - Ashani Kuttan
- James Graham Brown Cancer Center, Clinical Translational Research Building, University of Louisville, 505 South Hancock Street, Louisville, KY 40202
| | - John O. Trent
- James Graham Brown Cancer Center, Clinical Translational Research Building, University of Louisville, 505 South Hancock Street, Louisville, KY 40202
- Department of Medicine, University of Louisville, Clinical Translational Research Building, University of Louisville, 505 South Hancock Street, Louisville, KY 40202
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Abstract
This review summarizes the results of structural studies carried out with analogs of G-quadruplexes built from natural nucleotides. Several dozens of base-, sugar-, and phosphate derivatives of the biological building blocks have been incorporated into more than 50 potentially quadruplex forming DNA and RNA oligonucleotides and the stability and folding topology of the resultant intramolecular, bimolecular and tetramolecular architectures characterized. The TG4T, TG5T, the 15 nucleotide-long thrombin binding aptamer, and the human telomere repeat AG3(TTAG3)3 sequences were modified in most cases, and four guanine analogs can be noted as being particularly useful in structural studies. These are the fluorescent 2-aminopurine, the 8-bromo-, and 8-methylguanines, and the hypoxanthine. The latter three analogs stabilize a given fold in a mixture of structures making possible accurate structural determinations by circular dichroism and nuclear magnetic resonance measurements.
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Affiliation(s)
- Janos Sagi
- a Rimstone Laboratory , RLI, 29 Lancaster Way, Cheshire , CT , 06410 , USA
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19
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Le HT, Buscaglia R, Dean WL, Chaires JB, Trent JO. Calculation of hydrodynamic properties for G-quadruplex nucleic acid structures from in silico bead models. Top Curr Chem (Cham) 2013; 330:179-210. [PMID: 22886555 PMCID: PMC3580009 DOI: 10.1007/128_2012_351] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Nucleic acids enriched in guanine bases can adopt unique quadruple helical tertiary structures known as G-quadruplexes. G-quadruplexes have emerged as attractive drug targets as many G-quadruplex-forming sequences have been discovered in functionally critical sites within the human genome, including the telomere, oncogene promoters, and mRNA processing sites. A single G-quadruplex-forming sequence can adopt one of many folding topologies, often resulting in a lack of a single definitive atomic-level resolution structure for many of these sequences and a major challenge to the discovery of G-quadruplex-selective small molecule drugs. Low-resolution techniques employed to study G-quadruplex structures (e.g., CD spectroscopy) are often unable to discern between G-quadruplex structural ensembles, while high-resolution techniques (e.g., NMR spectroscopy) can be overwhelmed by a highly polymorphic system. Hydrodynamic bead modeling is an approach to studying G-quadruplex structures that could bridge the gap between low-resolution techniques and high-resolution molecular models. Here, we present a discussion of hydrodynamic bead modeling in the context of studying G-quadruplex structures, highlighting recent successes and limitations to this approach, as well as an example featuring a G-quadruplex structure formed from the human telomere. This example can easily be adapted to the investigation of any other G-quadruplex-forming sequences.
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Affiliation(s)
- Huy T Le
- Clinical and Translation Research Building, University of Louisville, 505 S. Hancock St, Louisville, KY, 40202, USA
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20
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Le HT, Miller MC, Buscaglia R, Dean WL, Holt PA, Chaires JB, Trent JO. Not all G-quadruplexes are created equally: an investigation of the structural polymorphism of the c-Myc G-quadruplex-forming sequence and its interaction with the porphyrin TMPyP4. Org Biomol Chem 2012; 10:9393-404. [PMID: 23108607 PMCID: PMC3501587 DOI: 10.1039/c2ob26504d] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III(1) promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of G-quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail.
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Affiliation(s)
- Huy T. Le
- Department of Biochemistry & Molecular Biology, School of Medicine, University of Louisville, HSC-A Building, Room 616 Louisville, Kentucky 40292; Phone: (502) 852-6221; Fax: (502) 852-6222
| | - M. Clarke Miller
- James G. Brown Cancer Center, University of Louisville, 529 South Jackson Street Louisville, KY 40202; Phone:(502) 562-4375
| | - Robert Buscaglia
- Department of Biochemistry & Molecular Biology, School of Medicine, University of Louisville, HSC-A Building, Room 616 Louisville, Kentucky 40292; Phone: (502) 852-6221; Fax: (502) 852-6222
| | - William L. Dean
- James G. Brown Cancer Center, University of Louisville, 529 South Jackson Street Louisville, KY 40202; Phone:(502) 562-4375
- Department of Medicine, School of Medicine, University of Louisville, 550 South Jackson Street, Louisville, KY 40202; Phone: (502) 852-5241; Fax: (502) 852-6233
| | - Patrick A. Holt
- Department of Biochemistry & Molecular Biology, School of Medicine, University of Louisville, HSC-A Building, Room 616 Louisville, Kentucky 40292; Phone: (502) 852-6221; Fax: (502) 852-6222
| | - Jonathan B. Chaires
- Department of Biochemistry & Molecular Biology, School of Medicine, University of Louisville, HSC-A Building, Room 616 Louisville, Kentucky 40292; Phone: (502) 852-6221; Fax: (502) 852-6222
- James G. Brown Cancer Center, University of Louisville, 529 South Jackson Street Louisville, KY 40202; Phone:(502) 562-4375
- Department of Medicine, School of Medicine, University of Louisville, 550 South Jackson Street, Louisville, KY 40202; Phone: (502) 852-5241; Fax: (502) 852-6233
| | - John O. Trent
- Department of Biochemistry & Molecular Biology, School of Medicine, University of Louisville, HSC-A Building, Room 616 Louisville, Kentucky 40292; Phone: (502) 852-6221; Fax: (502) 852-6222
- James G. Brown Cancer Center, University of Louisville, 529 South Jackson Street Louisville, KY 40202; Phone:(502) 562-4375
- Department of Medicine, School of Medicine, University of Louisville, 550 South Jackson Street, Louisville, KY 40202; Phone: (502) 852-5241; Fax: (502) 852-6233
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21
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Verhaart IEC, Aartsma-Rus A. The effect of 6-thioguanine on alternative splicing and antisense-mediated exon skipping treatment for duchenne muscular dystrophy. PLOS CURRENTS 2012; 4. [PMID: 23259153 PMCID: PMC3523663 DOI: 10.1371/currents.md.597d700f92eaa70de261ea0d91821377] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, exon skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated exon skipping levels. Recently, enhanced exon skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the exon skipping efficacy of 2’-O-methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of exon skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD exon skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required.
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Affiliation(s)
- Ingrid E C Verhaart
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
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22
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Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer. Pharmacol Ther 2012; 136:202-15. [PMID: 22850531 DOI: 10.1016/j.pharmthera.2012.07.011] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2012] [Accepted: 07/16/2012] [Indexed: 01/29/2023]
Abstract
Nucleic acid-based aptamers can be selected from combinatorial libraries of synthetic oligonucleotides to bind, with affinity and specificity similar to antibodies, a wide range of biomedically relevant targets. Compared to protein therapeutics, aptamers exhibit significant advantages in terms of size, non-immunogenicity and wide synthetic accessibility. Various chemical modifications have been introduced in the natural oligonucleotide backbone of aptamers in order to increase their half-life, as well as their pharmacological properties. Very effective alternative approaches, devised in order to improve both the aptamer activity and stability, were based on the design of polyvalent aptamers, able to establish multivalent interactions with the target: thus, multiple copies of an aptamer can be assembled on the same molecular- or nanomaterial-based scaffold. In the present review, the thrombin binding aptamers (TBAs) are analyzed as a model system to study multiple-aptamer constructs aimed at improving their anticoagulation activity in terms of binding to the target and stability to enzymatic degradation. Indeed - even if the large number of chemically modified TBAs investigated in the last 20 years has led to encouraging results - a significant progress has been obtained only recently with bivalent or engineered dendritic TBA aptamers, or assemblies of TBAs on nanoparticles and DNA nanostructures. Furthermore, the modulation of the aptamers activity by means of tailored drug-active reversal agents, especially in the field of anticoagulant aptamers, as well as the reversibility of the TBA activity through the use of antidotes, such as porphyrins, complementary oligonucleotides or of external stimuli, are discussed.
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23
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Wang J, Gu J, Leszczynski J. The electronic spectra and the H-bonding pattern of the sulfur and selenium substituted guanines. J Comput Chem 2012; 33:1587-93. [DOI: 10.1002/jcc.22991] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2012] [Revised: 03/07/2012] [Accepted: 03/18/2012] [Indexed: 11/07/2022]
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Román M, Cabaleiro T, Ochoa D, Novalbos J, Chaparro M, Gisbert JP, Abad-Santos F. Validation of a genotyping method for analysis of TPMT polymorphisms. Clin Ther 2012; 34:878-84. [PMID: 22421577 DOI: 10.1016/j.clinthera.2012.02.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/14/2012] [Indexed: 01/26/2023]
Abstract
BACKGROUND Thiopurine methyltransferase (TPMT) catalyzes the methylation of thiopurine drugs such as azathioprine and 6-mercaptopurine. Several mutations in the TPMT gene correlate with low enzyme activity and adverse effects such as myelotoxicity. Hence, genotyping TPMT makes it possible to identify patients at high risk for drug toxicity. OBJECTIVE The aim of this study was to validate a TPMT genotyping method by comparing it with a conventional polymerase chain reaction (PCR) approach. METHODS LightSNiP is a real-time PCR method for the detection of TPMT*2, *3B, and *3C without a sequencing step. We evaluated the frequencies of 3 TPMT alleles in 111 white adult patients by comparing genotyping by LightSNiP with conventional PCR (sequencing). RESULTS No differences were observed between conventional genotyping with sequencing and LightSNiP for *2, *3B, and *3C, suggesting the validity of this method. CONCLUSIONS Compared with the conventional PCR sequencing method, the data suggest that LightSNiP correctly detected the TPMT *2, *3B, and *3C in this select population.
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Affiliation(s)
- Manuel Román
- Clinical Pharmacology Service, Hospital Universitario de la Princesa, Instituto Teófilo Hernando, UAM, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain
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25
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Armstrong L, Sharif JA, Galloway P, McGrogan P, Bishop J, Russell RK. Evaluating the use of metabolite measurement in children receiving treatment with a thiopurine. Aliment Pharmacol Ther 2011; 34:1106-14. [PMID: 21929546 DOI: 10.1111/j.1365-2036.2011.04848.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Clinical response to thiopurine medication is related to the concentration of its metabolites. Proxy measures are traditionally used to assess dose adequacy. We present our experience of using tioguanine (previously known/formerly referred to as thioguanine) metabolite measurements in paediatric patients and evaluate their effect on clinical practice. AIMS To report our experience of using tioguanine metabolite measurements in paediatric patients and to evaluate their effects on clinical practice. METHODS The 6-tioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) were measured in children prescribed thiopurine medication for at least 3 months. Data were collected on thiopurine methyl transferase (TPMT) genotype, drug dose, laboratory indices and management changes. Therapeutic 6-TGN levels were defined as 235-400 pmol/8 × 10(8) RBCs. Seventy individuals (30 males) with a median age of 15 years. Underlying diagnoses were 'IBD' (68/70) and two cases of eosinophilic colitis. Sixty-three were treated with azathioprine and seven with mercaptopurine. A total of 103 separate measurements were made. RESULTS On initial measurement, 68% of patients had 6-TGN levels outside therapeutic levels despite standard thiopurine dosing. Initial 6-TGN levels were significantly higher in patients with TPMT mutations. Toxicity occurred in seven cases. The 6-TGN levels were significantly higher in those with signs of marrow toxicity. The 6-TGN level correlated with WBC, leukocyte count, mean corpuscular volume (MCV) and ΔMCV; however, the ability of each of these to predict therapeutic 6-TGN levels was poor. After initial measurement, management was changed in 25/70 cases (36%). CONCLUSIONS 6-TGN levels were therapeutic in a minority of those patients who were tested. Proxy measures perform poorly in predicting therapeutic 6-TGN levels. Measuring thiopurine metabolites is useful for dosage adjustment in children, and for the detection of potential toxicity.
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Affiliation(s)
- L Armstrong
- Department of Gastroenterology, Hepatology and Nutrition, Royal Hospital for Sick Children, Glasgow, UK.
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26
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Deepa P, Kolandaivel P, Senthilkumar K. Structural properties and the effect of interaction of alkali (Li+, Na+, K+) and alkaline earth (Be2+, Mg2+, Ca2+) metal cations with G and SG-tetrads. COMPUT THEOR CHEM 2011. [DOI: 10.1016/j.comptc.2011.07.012] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
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27
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Miller MC, Trent JO. Resolution of quadruplex polymorphism by size-exclusion chromatography. ACTA ACUST UNITED AC 2011; Chapter 17:Unit17.3. [PMID: 21638270 DOI: 10.1002/0471142700.nc1703s45] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
This unit describes a method for separation of quadruplex species formed from the same sequence via size-exclusion chromatography (SEC). Polymorphism is inherent to quadruplex formation, and even relatively simple quadruplex-forming sequences, such as the human telomere sequence d(GGG(TTAGGG)(3)), can form a myriad of possible configurations. HPLC, especially using reversed-phase and anion-exchange methods, has been a mainstay of nucleic acids research and purification for many decades. These methods have been applied for separation of individual quadruplex species formed in a mixture from the same parent sequence.
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Affiliation(s)
- M Clarke Miller
- James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA
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28
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Miller MC, Le HT, Dean WL, Holt PA, Chaires JB, Trent JO. Polymorphism and resolution of oncogene promoter quadruplex-forming sequences. Org Biomol Chem 2011; 9:7633-7. [PMID: 21938285 DOI: 10.1039/c1ob05891f] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
We report the separation of several quadruplex species formed by ten promoter sequences by Size Exclusion Chromatography (SEC). Modification at the 5' or 3' ends or in loop regions of quadruplex forming sequences has become the standard technique for dealing with quadruplex polymorphism. However, conformations produced employing this method or by other means of artificially shifting the equilibrium may not represent the species that are present in vivo. This method enables an unperturbed view of the structural polymorphism inherent to quadruplex formation. Separation via SEC facilitates studies on quadruplex structure and biophysical properties without the need for sequence modification.
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Affiliation(s)
- M Clarke Miller
- Department of Medicine, University of Louisville, Louisville, KY 40202, USA
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29
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Seela F, Ding P, Budow S. DNA Gold Nanoparticle Conjugates Incorporating Thiooxonucleosides: 7-Deaza-6-thio-2′-deoxyguanosine as Gold Surface Anchor. Bioconjug Chem 2011; 22:794-807. [DOI: 10.1021/bc200069j] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Frank Seela
- Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany
- Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany
| | - Ping Ding
- Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany
- Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany
| | - Simone Budow
- Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany
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Abstract
IMPORTANCE OF THE FIELD traditional immunosuppressants, including azathioprine, remain the mainstay of therapy in steroid dependent/refractory patients with inflammatory bowel diseases (IBD). The main limitations of its use are its side effects appearing in about a fifth of the patients, including myelosuppression and liver toxicity. Major complications occur in patients with low thiopurine-S-methyltransferase (TPMT) enzyme activity; however, the clinical relevance of these tests remains conflictive. AREAS COVERED IN THIS REVIEW in this review, the authors aim to summarize the new data regarding the relationship between the pharmacology of thiopurines and pathogenesis of adverse events. WHAT THE READER WILL GAIN readers will gain an understanding of the metabolism of thiopurines, side effect profile, pharmacological background of side effects, importance of metabolite monitoring, clinical relevance of inherited differences in drug metabolism and other conditions (e.g., concomitant use of allopurinol) which can modify enzyme activity. By gaining an understanding of the pharmacology and metabolism of thiopurines, clinicians will be able to optimize thiopurine therapy in IBD. TAKE HOME MESSAGE TPMT testing and metabolite monitoring are still not considered the standard of care, and clinicians will continue to choose the approach that best suits their clinical practice and patient needs. Regardless of what strategy is chosen, patients need to be carefully monitored and well informed about the potential risks.
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Affiliation(s)
- Pal Miheller
- Semmelweis University, 2nd Department of Medicine, Budapest, Koranyi, Hungary
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31
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Sannohe Y, Sugiyama H. Overview of formation of G-quadruplex structures. ACTA ACUST UNITED AC 2010; Chapter 17:Unit 17.2.1-17. [PMID: 20201027 DOI: 10.1002/0471142700.nc1702s40] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
There are many structures that can be adopted by nucleic acids other than the Watson-Crick duplex. In particular, a noncanonical four-stranded topology, called a G-quadruplex, is of great interest because of its roles in key biological processes such as the maintenance of telomeres and regulation of gene transcription. This review describes the condition for forming the G-quadruplex structure, G-quadruplex-forming sequences, and methods for studying the structures.
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de Graaf P, Vos RM, de Boer NHK, Sinjewel A, Jharap B, Mulder CJJ, van Bodegraven AA, Veldkamp AI. Limited stability of thiopurine metabolites in blood samples: relevant in research and clinical practise. J Chromatogr B Analyt Technol Biomed Life Sci 2010; 878:1437-42. [PMID: 20399153 DOI: 10.1016/j.jchromb.2010.03.004] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2009] [Revised: 03/01/2010] [Accepted: 03/04/2010] [Indexed: 01/27/2023]
Abstract
BACKGROUND Monitoring of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) is used to assess compliance and explain adverse reactions in IBD-patients. Correlations between dosage, metabolite concentrations and therapeutic efficacy or toxicity are contradictive. Research is complicated by analytical problems as matrices analyzed and analytical procedures vary widely. Moreover, stability of thiopurine metabolites is not well documented, yet pivotal for interpretation of analytical outcomes. Therefore, we prospectively investigated metabolite stability in blood samples under standard storage conditions. METHODS Stability at room temperature and refrigeration (22 degrees C, 4 degrees C) was investigated during 1 week and frozen samples (-20 degrees C, -80 degrees C) were analyzed during 6 months storage. Ten patient samples were analyzed for each study period. RESULTS Median 6-TGN concentrations on day 7 decreased significantly to 53% and 90% during storage at ambient temperature or refrigeration. Median 6-MMP concentrations on day 7 decreased significantly to 55% and 86%, respectively. Samples stored at -20 degrees C also showed significant decreases in both 6-TGN and 6-MMP in comparison with baseline values. At -80 degrees C, only 6-MMP showed a significant decrease in values compared to baseline. CONCLUSION The stability of thiopurine metabolites is clearly a limiting factor in studies investigating utilisation of TDM and correlations with therapeutic outcome in IBD-patients. This has to be accounted for in clinical practice and (multi-center) trials investigating thiopurine drugs.
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Affiliation(s)
- P de Graaf
- Clinical Pharmacology and Pharmacy, VU University Medical Centre, Amsterdam, The Netherlands.
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33
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Guanine analogues enhance antisense oligonucleotide-induced exon skipping in dystrophin gene in vitro and in vivo. Mol Ther 2010; 18:812-8. [PMID: 20087314 DOI: 10.1038/mt.2009.320] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Exon skipping has demonstrated great potential for treating Duchenne muscular dystrophy (DMD) and other diseases. We have developed a drug-screening system using C2C12 myoblasts expressing a reporter green fluorescent phosphate (GFP), with its reading frame disrupted by the insertion of a targeted dystrophin exon. A library of 2,000 compounds (Spectrum collection; Microsource Discovery System) was screened to identify drugs capable of skipping targeted dystrophin exons or enhancing the exon-skipping effect by specific antisense oligomers. The 6-thioguanine (6TG) was effective for inducing skipping of both human dystrophin exon 50 (hDysE50) and mouse dystrophin exon 23 (mDysE23) in the cell culture systems and increased exon skipping efficiency (more than threefolds) when used in combination with phosphorodiamidate morpholino oligomers (PMO) in both myoblasts and myotubes. Guanine and its analogues were unable to induce detectable skipping of exon 23 when used alone but enhanced PMO-induced exon skipping significantly (approximately two times) in the muscles of dystrophic mdx mouse in vivo. Our results demonstrate that small-molecule compounds could enhance specific exon skipping synergistically with antisense oligomers for experimental therapy to human diseases.
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Radiolabeling of thioguanine with 125I for diagnosis and therapy: in vitro and in vivo evaluation. J Radioanal Nucl Chem 2009. [DOI: 10.1007/s10967-009-0349-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Saneyoshi H, Mazzini S, Aviñó A, Portella G, González C, Orozco M, Marquez VE, Eritja R. Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer. Nucleic Acids Res 2009; 37:5589-601. [PMID: 19620215 PMCID: PMC2761269 DOI: 10.1093/nar/gkp598] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Modified thrombin-binding aptamers carrying 2′-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2′-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked South/syn-dG nucleoside produced aptamers with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syn-dG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations. We conclude that conformationally locked bicyclo[3.1.0]hexane nucleosides appear to be excellent tools for studying the role of key conformational parameters that are critical for the formation of a stable, antiparallel G-tetrad DNA structures.
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Affiliation(s)
- Hisao Saneyoshi
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Stefania Mazzini
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Anna Aviñó
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Guillem Portella
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Carlos González
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Modesto Orozco
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
| | - Víctor E. Marquez
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
- *To whom correspondence should be addressed. Tel: +34 93 4039942; Fax: +34 93 2045904;
| | - Ramon Eritja
- Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA, DISMA, Universita’ degli Studi di Milano. Via Celoria 2, I-20133 Milano, Italy, Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 15, E-08028 Barcelona, Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-08028 Barcelona and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Department of Biochemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Instituto de Química Física Rocasolano. CSIC, C/Serrano, 119, 28006 Madrid, Spain
- *To whom correspondence should be addressed. Tel: +34 93 4039942; Fax: +34 93 2045904;
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Villani G. Properties of the Thiobase Pairs Hydrogen Bridges: A Theoretical Study. J Phys Chem B 2009; 113:2128-34. [PMID: 19166279 DOI: 10.1021/jp807670f] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Giovanni Villani
- Istituto per i Processi Chimico-Fisici, IPCF-CNR, Via G. Moruzzi, 1, I-56124 Pisa, Italy
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37
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Lahoud G, Timoshchuk V, Lebedev A, Arar K, Hou YM, Gamper H. Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine. Nucleic Acids Res 2008; 36:6999-7008. [PMID: 18987000 PMCID: PMC2602760 DOI: 10.1093/nar/gkn797] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.
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Affiliation(s)
- Georges Lahoud
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
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38
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Tomasko M, Vorlícková M, Sagi J. Substitution of adenine for guanine in the quadruplex-forming human telomere DNA sequence G(3)(T(2)AG(3))(3). Biochimie 2008; 91:171-9. [PMID: 18852018 DOI: 10.1016/j.biochi.2008.07.012] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2008] [Accepted: 07/30/2008] [Indexed: 11/17/2022]
Abstract
We have studied the formation and structural properties of quadruplexes of the human telomeric DNA sequence G(3)(T(2)AG(3))(3) and related sequences in which each guanine base was replaced by an adenine base. None of these single base substitutions hindered the formation of antiparallel quadruplexes, as shown by circular dichroism, gel electrophoresis, and UV thermal stability measurements in NaCl solutions. Effect of substitution did differ, however, depending on the position of the substituted base. The A-for-G substitution in the middle quartet of the antiparallel basket scaffold led to the most distorted and least stable structures and these sequences preferred to form bimolecular quadruplexes. Unlike G(3)(T(2)AG(3))(3), no structural transitions were observed for the A-containing analogs of G(3)(T(2)AG(3))(3) when sodium ions were replaced by potassium ions. The basic quadruplex topology remained the same for all sequences studied in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may have biological relevance.
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Affiliation(s)
- Martin Tomasko
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
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39
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Mekmaysy CS, Petraccone L, Garbett NC, Ragazzon PA, Gray R, Trent JO, Chaires JB. Effect of O6-methylguanine on the stability of G-quadruplex DNA. J Am Chem Soc 2008; 130:6710-1. [PMID: 18447358 DOI: 10.1021/ja801976h] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The effects of substitution of O6-methylguanine on the structure and stability of a human telomere quadruplex was studied by circular dichroism, thermal denaturation, analytical ultracentrifugation, and molecular dynamics simulations. The results show that, while quadruplex structures can form containing the modified base, they are much less stable than the normal unmodified structure. The extent of destabilization is critically dependent on the exact position of the modified base within the quadruplex structure.
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Affiliation(s)
- Chongkham S Mekmaysy
- James Graham Brown Cancer Center and University of Louisville, 529 South Jackson Street, Louisville, Kentucky 40202, USA
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40
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de Boer NKH, van Bodegraven AA, Jharap B, de Graaf P, Mulder CJJ. Drug Insight: pharmacology and toxicity of thiopurine therapy in patients with IBD. ACTA ACUST UNITED AC 2007; 4:686-94. [DOI: 10.1038/ncpgasthep1000] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2007] [Accepted: 09/25/2007] [Indexed: 02/08/2023]
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Sponer J, Spacková N. Molecular dynamics simulations and their application to four-stranded DNA. Methods 2007; 43:278-90. [PMID: 17967698 PMCID: PMC2431124 DOI: 10.1016/j.ymeth.2007.02.004] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2007] [Accepted: 02/14/2007] [Indexed: 11/30/2022] Open
Abstract
This review provides a critical assessment of the advantages and limitations of modeling methods available for guanine quadruplex (G-DNA) molecules. We characterize the relations of simulations to the experimental techniques and explain the actual meaning and significance of the results. The following aspects are discussed: pair-additive approximation of the empirical force fields, sampling limitations stemming from the simulation time and accuracy of description of base stacking, H-bonding, sugar-phosphate backbone and ions by force fields. Several methodological approaches complementing the classical explicit solvent molecular dynamics simulations are commented on, including enhanced sampling methods, continuum solvent methods, free energy calculations and gas phase simulations. The successes and pitfalls of recent simulation studies of G-DNA are demonstrated on selected results, including studies of cation interactions and dynamics of G-DNA stems, studies of base substitutions (inosine, thioguanine and mixed tetrads), analysis of possible kinetic intermediates in folding pathway of a G-DNA stem and analysis of loop regions of G-DNA molecules.
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Affiliation(s)
- Jirí Sponer
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
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42
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Affiliation(s)
- Mark Lukin
- Department of Pharmacological Sciences, State University of New York at Stony Brook, School of Medicine, 11794-8651, USA
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43
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Frohman EM, Havrdova E, Levinson B, Slanar O. Azathioprine myelosuppression in multiple sclerosis: characterizing thiopurine methyltransferase polymorphisms. Mult Scler 2006; 12:108-11. [PMID: 16459728 DOI: 10.1191/135248506ms1249cr] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
We describe two multiple sclerosis patients who developed pancytopenia following treatment with azathioprine. They were found to have the homozygous polymorphism for thiopurine methyltransferase deficiency and recovered after cessation of drug therapy. We review the literature concerning this molecular derangement and underscore the importance of performing surveillance testing for allelic characterization prior to treatment intervention with this agent for immune-mediated disorders.
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Affiliation(s)
- E M Frohman
- Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA.
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44
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López de la Osa J, González C, Gargallo R, Rueda M, Cubero E, Orozco M, Aviñó A, Eritja R. Destabilization of Quadruplex DNA by 8-Aminoguanine. Chembiochem 2005; 7:46-8. [PMID: 16292787 DOI: 10.1002/cbic.200500281] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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Breen DP, Marinaki AM, Arenas M, Hayes PC. Pharmacogenetic association with adverse drug reactions to azathioprine immunosuppressive therapy following liver transplantation. Liver Transpl 2005; 11:826-833. [PMID: 15973722 DOI: 10.1002/lt.20377] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Azathioprine (AZA) is a thiopurine prodrug commonly used in triple-immunosuppressive therapy following liver transplantation. Approximately 1 in 10 patients suffers side effects in response to the drug, the most problematic being bone marrow toxicity. There is evidence that polymorphisms in the genes encoding thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPase) predict adverse drug reactions to AZA therapy. Furthermore, common genetic polymorphisms in the gene encoding methylenetetrahydrofolate reductase (MTHFR) may have an indirect impact on thiopurine drug methylation by influencing levels of the methyl donor S-adenosylmethionine (SAM). The aim of this study was to determine whether polymorphisms in these candidate pharmacogenetic loci predict adverse drug reactions to AZA immunosuppressive therapy in liver transplant patients. A series of 65 liver transplant recipients were recruited to the study from the Liver Transplant Out-Patient clinic at The Royal Infirmary of Edinburgh. Clinical response to AZA was retrospectively correlated against TPMT activity, TPMT*2, *3A, and *3A genotypes, inosine triphosphatase (ITPA) 94C>A and IVS2+21A>C genotypes, and MTHFR 677C>T and 1298A>C genotypes. Variant TPMT, ITPA, and MTHFR genotypes were not significantly associated with adverse drug reactions to AZA, including bone marrow suppression. However, the 2 patients who suffered nodular regenerative hyperplasia (NRH) were both heterozygous for the TPMT*3A mutation. In conclusion, our findings suggest that TPMT, ITPA, and MTHFR genotypes do not predict adverse drug reactions, including bone marrow suppression, in liver transplant patients. However, the possible association between NRH and a heterozygous TPMT genotype should be investigated further.
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Affiliation(s)
- David P Breen
- College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK
| | | | - Monica Arenas
- Purine Research Laboratory, Guy's and St Thomas's Hospital, London, UK
| | - Peter C Hayes
- Scottish Liver Transplant Unit, The Royal Infirmary of Edinburgh at Little France, Edinburgh, UK
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Abstract
The incorporation of 6-thioguanine (S6G) into DNA is a prerequisite for its cytotoxic action, but duplex structure is not significantly perturbed by the presence of the lesion [J. Bohon and C. R. de los Santos (2003) Nucleic Acids Res., 31, 1331–1338]. It is therefore possible that the mechanism of cytotoxicity relies on a loss of stability rather than a pathway involving direct structural recognition. The research described here focuses on the changes in thermodynamic properties of duplex DNA owing to the introduction of S6G as well as the kinetic properties of base pairs involving S6G. Replacement of a guanine in a G•C pair by S6G results in ∼1 kcal/mol less favorable Gibbs free energy of duplex formation at 37°C. S6G•T and G•T mismatch-containing duplexes have almost identical Gibbs free energy at 37°C, with values ∼3 kcal/mol less favorable than that of the control. Base pair stability is affected by S6G. The lifetime of the normal G•C base pair is ∼125 ms, whereas that of the G•T mismatch is below the detection limit. The lifetimes of S6G•C and S6G•T pairs are ∼7 and 2 ms, respectively, demonstrating that, although S6G significantly decreases the stability of the pairing with cytosine, it slightly increases that of a mismatch.
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47
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Meng F, Xu W, Liu C. Theoretical study of incorporating 6-thioguanine into a guanine tetrad and their influence on the metal ion–guanine tetrad. Chem Phys Lett 2004. [DOI: 10.1016/j.cplett.2004.03.128] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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Bohon J, de los Santos CR. Structural effect of the anticancer agent 6-thioguanine on duplex DNA. Nucleic Acids Res 2003; 31:1331-8. [PMID: 12582253 PMCID: PMC150222 DOI: 10.1093/nar/gkg203] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G.T) and opposite cytosine (S6G.C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson-Crick hydrogen bonding in place. The S6G.T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.
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Affiliation(s)
- Jen Bohon
- Department of Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA
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49
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Somerville L, Krynetski EY, Krynetskaia NF, Beger RD, Zhang W, Marhefka CA, Evans WE, Kriwacki RW. Structure and dynamics of thioguanine-modified duplex DNA. J Biol Chem 2003; 278:1005-11. [PMID: 12401802 DOI: 10.1074/jbc.m204243200] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.
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Affiliation(s)
- Lilla Somerville
- Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA
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Jaumot J, Escaja N, Gargallo R, González C, Pedroso E, Tauler R. Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids. Nucleic Acids Res 2002; 30:e92. [PMID: 12202780 PMCID: PMC137437 DOI: 10.1093/nar/gnf091] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d<pTGCTCGCT>. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl2 by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0 degrees C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration.
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Affiliation(s)
- Joaquim Jaumot
- Departament de Química Analítica, Universitat de Barcelona, Diagonal 647, E-08028, Barcelona, Spain
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