Anna Aviñó1, Stefania Mazzini2, Carme Fàbrega3, Pablo Peñalver4, Raimundo Gargallo5, Juan Carlos Morales4, Ramon Eritja6. 1. Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain. Electronic address: aaagma@cid.csic.es. 2. Department of Food, Environmental and Nutritional Sciences (DEFENS), Section of Chemical and Biomolecular Sciences, University of Milan, Via Celoria 2, 20133 Milan, Italy. 3. Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain. 4. Department of Biochemistry and Molecular Pharmacology, Instituto de Parasitología y Biomedicina, CSIC, Parque Tecnológico Ciencias de la Salud, Avda. del Conocimiento, s/n, 18016 Armilla, Granada, Spain; Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC - Universidad de Sevilla, Américo Vespucio 49, 41092 Sevilla, Spain. 5. Department of Chemical Engineering and Analytical Chemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Spain. 6. Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain. Electronic address: recgma@cid.csic.es.
Abstract
BACKGROUND: Guanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2'-deoxyguanosine. METHODS: Modified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. RESULTS: The most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. CONCLUSIONS: The substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. GENERAL SIGNIFICANCE: Modified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
BACKGROUND:Guanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2'-deoxyguanosine. METHODS: Modified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. RESULTS: The most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. CONCLUSIONS: The substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. GENERAL SIGNIFICANCE: Modified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.