Using circular dichroism spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we studied conformational properties of guanine-rich DNA strands of the fragile X chromosome repeats d(GGC)n, d(GCG)n and d(CGG)n, with n = 2, 4, 8 and 16. These strands are generally considered in the literature to form guanine tetraplexes responsible for the repeat expansion. However, we show in this paper that the repeats are reluctant to form tetraplexes. At physiological concentrations of either Na+ or K+ ions, the hexamers and dodecamers associate to form homoduplexes and the longer repeats generate homoduplexes and hairpins. The tetraplexes are rarely observed being relatively most stable with d(GGC)n and least stable with d(GCG)n. The tetraplexes are exclusively formed in the presence of K+ ions, at salt concentrations higher than physiological, more easily at higher than physiological temperatures, and they arise with extremely long kinetics (even days). Moreover, the capability to form tetraplexes sharply diminishes with the oligonucleotide length. These facts make the concept of the tetraplex appearance in this motif in vivo very improbable. Rather, a hairpin of the fragile X repeats, whose stability increases with the repeat length, is the probable structure responsible for the repeat expansion in genomes.

• MeSH
• Circular Dichroism MeSH
• DNA chemistry   drug effects   genetics   metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• G-Quadruplexes MeSH
• Guanine metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation * drug effects   MeSH
• Humans MeSH
• Chromosomes, Human, X genetics   MeSH
• Oligodeoxyribonucleotides chemistry   genetics   metabolism   MeSH
• Osmolar Concentration MeSH
• Base Sequence MeSH
• Salts pharmacology   MeSH
• Spectrophotometry, Ultraviolet MeSH
• Fragile X Syndrome genetics   MeSH
• Thermodynamics MeSH
• Trinucleotide Repeats genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Guanine MeSH
• Oligodeoxyribonucleotides MeSH
• Salts MeSH

UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)(n) (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)(n) strand is wider than has been shown so far. (GCC)(n) strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C.C(+) pairs. The (GCC)(16) strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)(n) strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and monomolecular with (GCC)(16). i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)(n) strand duplex with a complementary (GGC)(n) strand.

• MeSH
• Circular Dichroism MeSH
• Dimerization MeSH
• DNA chemistry   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Spectrophotometry, Ultraviolet MeSH
• Fragile X Syndrome genetics   MeSH
• Trinucleotide Repeats * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH

CD spectroscopy and PAGE were used to cooperatively analyze melting conformers of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10 formed a homoduplex in neutral solutions containing physiological concentrations of salts and this homoduplex was not destabilized even in the terminal (GA)3 hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this oligonucleotide preserved the conformation adopted by (TA)10. This observation demonstrates that homoduplexes of alternating GA and TA sequences can co-exist in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them reversibly. The concentration of salt controlled the conformational switching. Hence, guanine and thymine share significant properties regarding complementarity to adenine, while the TA and GA sequences can stack in at least two mutually compatible ways within the DNA duplexes analyzed here. These properties extend our knowledge of non-canonical structures of DNA.

• MeSH
• Circular Dichroism MeSH
• Dinucleotide Repeats * MeSH
• DNA chemistry   genetics   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Nucleic Acid Heteroduplexes chemistry   genetics   MeSH
• Nucleic Acid Conformation * MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Nucleic Acid Heteroduplexes MeSH

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.

• MeSH
• Biophysics MeSH
• Biophysical Phenomena MeSH
• Circular Dichroism MeSH
• DNA chemistry   MeSH
• Glycols pharmacology   MeSH
• Nucleic Acid Conformation drug effects   MeSH
• Crystallization MeSH
• Oligodeoxyribonucleotides chemistry   MeSH
• Solutions MeSH
• Base Sequence MeSH
• In Vitro Techniques MeSH
• Water MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DNA MeSH
• Glycols MeSH
• hexylene glycol MeSH Browser
• Oligodeoxyribonucleotides MeSH
• Solutions MeSH
• Water MeSH