Energetic aspects of locked nucleic acids quadruplex association and dissociation

178

210

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5′ end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Section: Resultsmentioning

confidence: 99%

Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes

Gros

Rosu

Amrane

et al. 2007

178

210

“…The TDS in K + and Na + buffers are shown in Figure 1C and D. These TDS spectra were obtained by difference between the absorbance spectra recorded above and below the observed transition. They exhibit the typical pattern of a G-quadruplex structure with two positive maxima at 240 and 275 nm and a negative minimum around 295 nm (26,33,37). Furthermore, the CD spectra of these structures were in agreement with the formation of quadruplexes (Figure 1E and F) (38,39).…”

Section: Resultsmentioning

confidence: 99%

Stability of intramolecular quadruplexes: sequence effects in the central loop

Guédin¹,

Alberti²,

Mergny³

2009

Hundreds of thousands of putative quadruplex sequences have been found in the human genome. It is important to understand the rules that govern the stability of these intramolecular structures. In this report, we analysed sequence effects in a 3-base-long central loop, keeping the rest of the quadruplex unchanged. A first series of 36 different sequences were compared; they correspond to the general formula GGGTTTGGGHNHGGGTTTGGG. One clear rule emerged from the comparison of all sequence motifs: the presence of an adenine at the first position of the loop was significantly detrimental to stability. In contrast, adenines have no detrimental effect when present at the second or third position of the loop. Cytosines may either have a stabilizing or destabilizing effect depending on their position. In general, the correlation between the Tm or ΔG° in sodium and potassium was weak. To determine if these sequence effects could be generalized to different quadruplexes, specific loops were tested in different sequence contexts. Analysis of 26 extra sequences confirmed the general destabilizing effect of adenine as the first base of the loop(s). Finally, analysis of some of the sequences by microcalorimetry (DSC) confirmed the differences found between the sequence motifs.

“…More recently, we (34,35) and others (36) analyzed the kinetics of quadruplex formation with short sequences. From a kinetic point of view, the association reaction strongly depends on strand concentration with an experimentally determined order approaching four (33,34,36); an exception being the four-stranded structures formed by the sugar-modified oligonucleotides known as locked nucleic acids (37). Second, the kinetic inertia of these complexes, rather than being a hindrance, facilitates the study of the uncoupling of the folding and unfolding phenomenon.…”

Section: Introductionmentioning

confidence: 99%

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation

Cian¹,

Mergny²

2007

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, koff) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, kon. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on kon. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.