Duplex Dissociation of Telomere DNAs Induced by Molecular Crowding

Abstract

Because of the importance of telomere DNAs, the structures of these DNAs in vivo are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. To understand the structure of biomolecules in vivo, their properties studied in vitro are extrapolated to the in vivo condition, while the condition in a living cell is inherently molecularly crowded and a nonideal solution contains various biomolecules. We investigated the effect of molecular crowding, which is one of the most important cellular environmental conditions, on the structure and stability of the telomere and G-rich and C-rich DNAs using circular dichroism (CD) spectra, CD melting curves, and isothermal titration calorimetry (ITC). The CD spectra and CD melting curves of G-rich DNA, C-rich DNA, and the 1:1 mixture of G-rich and C-rich DNAs showed that each G-rich DNA, C-rich DNA, and the 1:1 mixture form the antiparallel G-quadruplex, I-motif, and duplex, respectively, in the noncrowding condition as previously considered. On the contrary, the G-rich and C-rich DNAs individually form the parallel G-quadruplex and I-motif, respectively, in the molecular crowding condition, and the 1:1 mixture folds into the parallel G-quadruplex and I-motif but does not form a duplex. The ITC measurements indicated that the thermodynamic stability (DeltaG degrees (20)) of the duplex formation between the G-rich and C-rich DNAs in the noncrowding condition was -10.2 kcal mol(-)(1), while only a small heat change was observed in the ITC measurements in the molecular crowding condition. These ITC results also demonstrated that the molecular crowding condition prevents any duplex formation between G-rich and C-rich DNAs. These results indicate that a structural polymorphism of the telomere DNAs is induced by molecular crowding in vivo.

Parts-per-Million of Polyethylene Glycol as a Non-Interfering Blocking Agent for Homogeneous Biosensor Development

“…25,26 We recently reported the use of PEG as a stabilizer for nanoparticles, [27][28][29] and PEG is also a commonly used artificial crowding agent. [30][31][32] With the common perception that PEG is an anti-fouling molecule, unmodified PEG appears to be a poor blocker. In this Technical Note, we report that very diluted and unmodified PEGs can effectively block plastic surfaces for homogeneous assays involving DNAs, proteins or lipids ( Figure 1E).…”

Targeting G-Quadruplex DNA with Small Molecules

“…The equilibrium between duplex and G-quadruplex/i-motif structures can be influenced by DNA binding proteins, [7] small-molecule binding, [10][11][12] negative supercoiling, [12][13][14][15][16] changes in pH and temperature, [29] and molecular crowding. [30] plex-specific antibodies. [75] Subsequent studies using a combination of RNAi and antibodies have provided a direct link between telomeric end-binding proteins and the formation of G-quadruplex structures in ciliates.…”

“…[45] These results highlight the possibility that i-motifs might be formed under physiological conditions in vivo through molecular binding and/or crowding interactions. [30] While direct and indirect evidence supports the presence and biological relevance of G-quadruplex structures located in the 3' single-stranded overhangs of ciliate telomeres in vivo (presented in Section 4), much less information is available regarding the potential relevance of the equilibrium depicted in Fig. 3, where certain G-C rich DNA sequences can exist as a mixture of G-quadruplex/i-motif and canonical duplex DNA in vitro.…”

“…2). [20][21][22][23][24][25][26] Two of the 26 (or more) examples of intramolecular G-quadruplex topologies are shown in Fig 2. [21,27] While Gquadruplexes can exhibit thermodynamic stabilities comparable to those of the corresponding duplex structures, [19,[28][29][30] the presence and function(s) of these structures in vivo remains an open question. Intermolecular G-quadruplex structures have been proposed as intermediates or precursors of recombination and/or viral integration, [23,31,32] while intramolecular Gquadruplexes have been implicated in the regulation of gene expression and chromosome stability.…”

“…3, where certain G-C rich DNA sequences can exist as a mixture of G-quadruplex/i-motif and canonical duplex DNA in vitro. [29,30] Cell-permeable ligands that selectively bind to one or more of these structures might provide a means for probing the existence and/or controlling the function(s) associated with these structures. These same interactions might also provide a new source of therapeutic agents and targets.…”