Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes†

doi:10.7503/cjcu20140408

Abstract

Abstract:

G-rich tandem repeats sequences are able to fold into structures called G-quadruplexes(G4s). G4s have complicated topological structures, which closely depend on the sequences and the solution conditions. In this work, the effects of loops, flanking bases and monovalent cations on the structure of G4s were investigated by gel electrophoresis, circular dichroism spectra and UV-Melting. The results showed that G4 forming sequences with short loops were favorable to form parallel structure in K⁺ solution, and the parallel G4s without flanking bases were able to further form G4 multimers, but the anti-parallel or mixed parallel/anti-parallel G4s were not. The monovalent cations had an influence on the adopted structures; K⁺ was more favorable for the formation of parallel G4 and G4 multimers than N $H 4 +$ and Na⁺. The addition of non-guanine bases to the terminals of parallel G4s, or the transformation of the parallel G4s to non-parallel structures by changing the cations, could inhibit the formation of G4 multimers. Furthermore, loop length could affect the thermo-stability of G4s; G4s with short loops had higher thermo-stability. The G4 multimers showed lower thermo-stability than G4 monomer, and their size and amount became smaller with the temperature increase. These results suggest that G4s with short loops and no flanking bases fold into intramolecular parallel structures firstly, and then form multimers through end stacking. The flanking bases and loops located on the G-quartets at 3' or 5' terminal hinder the stacking interactions, and inhibit the multimer assembly. Our results may offer useful clues for the structure and function studies of G4s.

Key words: G-Quadruplex, Topological structure, DNA, Self-assembly, Multimer

CLC Number:

O629.74

TrendMD:

CHANG Tianjun, LI Gang, ZHAO Kexu, BAN Lin, BIAN Wenxiu, BING Tao, SHANGGUAN Dihua. Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes^†[J]. Chem. J. Chinese Universities, 2014, 35(12): 2556.

Figures/Tables 5

Fig.1 Schematic representation of G-quartet and G-quadruplex structures (A) Chemical structure of G-quartet; schematic of intramolecular parallel G-quadruplex(B), intramolecular antiparallel G-quadruplex(C), mixed intramolecular parallel/antiparallel G-quadruplex(D), bimolecular antiparallel G-quadruplex(E), tetramolecular parallel G-quadruplex(F), dimeric parallel G-quadruplex of c-kit2[18](G) and dimeric parallel G-quadruplexes with 5'-5' stacking motif[19](H).

Table 1 DNA sequences and their Tm values in 20 mmol/L KCl*

Fig.2 CD spectra of DNAs without flanking base in K+ solution Sequences from a to i: VEGF18, EAD15, c-Myc16,c-Myc(b)20, c-kit19, CEB23, Bcl-2, HT, ILPR.

Fig.3 DNA analysis by denaturing-PAGE(A) and native-PAGE(B) (A) The gels for denaturing-PAGE contains 7 mol/L urea; (B) both the gels and the running buffers contain 20 mmol/L KCl for native-PAGE. The sequences were indicated on top of the gels. The horizontal dashed lines indicate the position of the molecular weight markers(Mw). The Mw of the sequences are: 15-mer,(AAGT)3AAG; 20-mer,(AAGT)5; 30-mer, (AAGT)7AA; 40-mer,(AAGT)10.

Fig.4 Native-PAGE of G-quadruplex forming sequences without flanking bases folded in buffers containing various concentrations of K+(A), Na+(B) and NH4+(C) Both the gels and running buffers contain 20 mmol/L K+, Na+, and N H 4 + ,respectively. (A1—C1) EAD15; (A2—C2) c-Myc16; (A3—C3) VEGF18; (A4—C4) c-kit19; (A5—C5) c-Myc(b)20; (A6—C6) CEB23. Concentration/(mmol·L-1): a. 20; b. 50; c. 80; d. 100.

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