Structural characterization of quadruplex DNA with in-cell EPR approaches

Abstract

Guanosine-rich DNA sequences have the potential to adopt four-stranded conformations termed quadruplexes. The chromosomes of higher organisms are capped by so-called telomeres that are composed of repeats of the sequence TTAGGG. Up to 200 nucleotides of the G-rich strand form an overhang that is suspected to fold into intramolecular G-quadruplexes. Since induction of quadruplexes at the telomeres results in anti-proliferative effects, the intracellular structure of G-quadruplexes is of high interest as an anti-cancer drug target. Here we give a perspective on the elucidation of DNA sequence folds by electron paramagnetic resonance (EPR) distance measurements. The technique complements X-ray crystallography and NMR spectroscopy, as it can be applied in noncrystalline states, is not intrinsically limited by the size of the bio-macromolecular complex, and is able to analyze flexible structures or coexisting DNA conformation.

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

“…[61] Although EPR spectroscopy is more sensitive than NMR spectroscopy, an extraordinarily high concentration of the target (50-250 mm) is needed for both methods to obtain the necessary sensitivity (Table 1). [23,[62][63][64] 3. Cellular Systems for In-Cell NMR and EPR Spectroscopy and Their Applications…”

“…The E. coli in-cell system requires intracellular expression of the macromolecule of interest in the presence of a suitable isotopically labeled medium. [65] To eliminate signal contribu- information conformations [12,15,66,67,78,98,99] dynamics [36,76] interactions [11, 16-18, 34, 35, 70, 73, 74, 87, 111] posttranslational modifications [10,31,86,87,[112][113][114][115] conformations [22,37,38] interactions [22,37,38] distances [23,24,63,64,101] conformations [23,24,63,64,101] interactions dynamics . tions of leaked metabolites and target molecules, cells are subjected to extensive washing steps prior to the acquisition of the in-cell NMR spectrum.…”

Nanometal Surface Energy Transfer Optical Ruler for Measuring a Human Telomere Structure

“…Telomeres are one example of many in the genome where quadruplex-protein interaction is coupled to genetic function (5)(6)(7)(8)(9)(10)(11). The desire to gain structural insight into the possible folding landscapes of guanine repeats led the experimental focus towards telomeric G-quadruplexes, which have been observed to preferentially form (3 + 1) hybrid antiparallel Hybrid-1 and Hybrid-2 structures (12)(13)(14)(15)(16)(17)(18). Several biophysical methods have been employed to investigate the folding of these structures.…”

“…NMR, for instance, is used for short contact measurements to deduce a global structure but requires highly concentrated samples and produces broadened line widths at high salt concentrations (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). EPR is perhaps the most promising method for observing the G-quadruplex folding event at low concentration, but is incapable of measurements at room temperature (12,13,25). Optical methods, like circular dichroism, are effective in recognizing the presence of a folded quadruplex, but fail to distinguish between the more relevant (3 + 1) hybrid antiparallel structures in telomeres (26)(27)(28)(29)(30).…”

Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd3+-DOTA

“…[14][15][16] Similarly, EPR has been used to detect the formation of both duplex and triplex DNA structures. [14][15][16] Similarly, EPR has been used to detect the formation of both duplex and triplex DNA structures.…”