Biophysical properties of quadruple helices of modified human telomeric DNA

Abstract

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. The guanine rich strand can fold into four-stranded G-quadruplex structures, which have recently become attractive for biomedical research. Indeed, the aptamers based on the quadruplex motif may prove useful as tools aimed at binding and inhibiting particular proteins, catalyzing various biochemical reactions, or even serving as pharmaceutically active agents. The incorporation of modified bases into oligonucleotides can have profound effects on their folding and may produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the adenines of the human telomeric repeat oligonucleotide d(TAGGGT) and d(AGGGT) were substituted by 2'-deoxy-8-(propyn-1-yl)adenosine (A-->APr) or by 8-bromodeoxyadenosine (A-->ABr). The biophysical properties of the resulting quadruplex structures were compared with the unmodified quadruplexes. NMR and CD spectra of the studied sequences were characteristic of parallel-stranded, tetramolecular quadruplexes. The analysis of the equilibrium melting curves reveals that the modifications stabilize the quadruplex structure. The results are useful when considering the design of novel aptameric nucleic acids with diverse molecular recognition capabilities that would not be present using native RNA/DNA sequences.

Energetic aspects of locked nucleic acids quadruplex association and dissociation

“…This arises when the systems are not at thermodynamic equilibrium during the temperature changes (the scan rate used was 1.08C/min) and it is due to slow rates of dissociation or association process, indicating that these curves do not correspond to the true equilibrium curves. 17,21,33 To obtain reliable thermodynamic data, the equilibrium melting curves were derived following the procedure described in ''Materials and Methods'' section. The melting curves were obtained reporting CD signal at 263 nm as function of temperature ( Figure 3).…”

“…15 Different modifications have been proposed involving the base, the phosphate group or the sugar-phosphate backbone in an attempt to stabilize quadruplex structure. [16][17][18][19][20][21] For example, it was demonstrated that a nucleic acids analogs such as peptidic nucleic acids (PNAs) can form PNA/DNA hybrid quadruplexes. 19 Unfortunately, the poor water solubility of the all PNA oligomers limits their potential applications.…”

“…The CD measurements were performed at constant single strand concentration of 2 Â 10 À4 M. The equilibrium melting curves were recorded in the range 5-1058C using a temperature step of 58C following a procedure previously reported. 18,21 The samples were incubated at each temperature to achieve the equilibrium before recording the CD. The equilibrium was established by waiting until no changes were observed with time.…”

Physico-chemical analysis of G-quadruplex containing bunch-oligonucleotides

“…3): (i) the heating curve shows a biphasic profile; (ii) in the cooling the CD signal is recovered in the range of temperature 105-75 • C; (iii) an hysteresis is present between melting and annealing process in the range of temperature 75-20 • C. These findings reveal the presence of a partially kinetically controlled complex melting process. Hence, to avoid kinetic control in the denaturation/renaturation processes and assure the equilibrium achievement, the CD melting curve was obtained in equilibrium conditions following a procedure previously described [39,40]. The sample was incubated at each temperature for a suitable time to achieve the equilibrium before recording the CD spectrum.…”

“…The equilibrium-melting curve was recorded in the range 15-105 • C using a temperature step of 5 • C following a previously reported procedure [39,40]. The sample was incubated at each temperature for a suitable time to achieve the equilibrium before recording the CD signal.…”