G-Quadruplex and i-Motif Are Mutually Exclusive in ILPR Double-Stranded DNA (original) (raw)
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Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex
2021
ABSTRACTG-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While many in-vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in-vivo in double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than...
Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex
Nucleic Acids Research, 2021
G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics wa...
Journal of the American Chemical Society, 2010
Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double stranded DNA. Whereas single molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C) rich region complementary to G quadruplex forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5′-(TGTCCCCACACCCC) 2 , a predominate variant in the insulin linked polymorphic region (ILPR). We have observed two species with the change in contour length (ΔL) of 10.4 (±0.1) and 5.1 (±0.5) nm, respectively. Since ΔL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br 2 footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23 % to 1.3 %) with pH 5.5 7.0, which is a well established behavior for imotifs. In contrast to the i-motif, the formation of the second species with ΔL of 5.1 nm remains unchanged (6.1±1.6 %) in the same pH range, implying that pH sensitive C:CH + pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the ΔG unfold of imotif (16.0 ±0.8 kcal/mol), the decreased free energy in the partially folded structure (ΔG unfold 10.4 ± 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH + pairs. Both ΔL and ΔG unfold argue for the intermediate nature of the partially folded structure in comparison to the imotif. In line with this argument, we have directly observed the unfolding of i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.
G-quadruplex unfolding in higher-order DNA structures
Chemical Communications, 2013
G-quadruplex unfolding within a sequence of two quadruplex units was characterized by gel electrophoresis, calorimetry and spectroscopy. The obtained results suggest that the kinetics and thermodynamics of the individual quadruplex unfolding are affected by its interaction with other DNA secondary structural elements.
Chemistry - A European Journal, 2013
Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate-DNA conjugates containing the oligonucleotide sequences of Gquadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose-TBA conjugate shows stacking interactions between the carbohydrate and the DNA G-tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5'-end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G-quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G-quadruplex ligands.
Biophysical Journal, 2013
The structure of bio-molecules such as DNA and RNA depends strongly on the cation binding sites. Epigenetic regulation mechanism may depend in part on the structure of DNA and its ability to interact with regulatory proteins. Understanding of the dynamic role of cations on DNA structure and how they get affected by several modifications involved in chromatin remodeling will give light on how exactly these mechanisms control such complicated scenarios. Nevertheless, even the location and effect of the cations on DNA or RNA structure on simple systems (protein free) is not clearly understood or predictable, or how well we can model such systems with the current available modeling methods. We choose a model system of G-quadruplex DNA, molecules well known to depend for its structures strongly on the cations present, to study the effect of the cation location and number in the modeling of these structures in order to evaluate how well molecular dynamics (CHARMM) performs.
Single Molecule Force Spectroscopy on G-Quadruplex DNA
Chemistry - A European Journal, 2009
Interest in guanine (G) quadruplexes[1] has intensified over the past decade because they are found in human telomeres,[2] the chromosome ends that govern gene stability, and other parts of the genome, especially in promoters.[3] They have been shown to inhibit the activity of telomerase, an enzyme which maintains the proper length of telomere DNAs and is overexpressed in many types of cancer.[4] As such, the G-quadruplex DNA has been an attractive target for cancer therapy.[5] Despite the wide interest in the topology of G-quadruplexes, their physical properties are not well-understood.[6] There are large differences between the kinetic data obtained in solution using fluorescence and on surfaces using surface plasmon resonance (SPR) approaches,[7] possibly due to destabilization by fluorophore labelling[7a-c] and surface hindrance in the indirect hybridization based SPR approach.[7d] The kinetics and thermodynamics are expected to be different for G-quadruplexes formed from uni-, bi-, and tetra-molecular DNA strands. Furthermore, G-quadruplexes formed from a single DNA strand may have different conformations,[6.7] where minor changes in the DNA sequence may result in significant differences in their thermodynamic properties.[6] By taking measurements directly on unlabelled individual single molecules, one at a time, avoiding ensemble averaging, single-molecule (SM) force spectroscopy (FS) based on atomic force microscopy (AFM) has been demonstrated as a powerful tool to gain insights to a wide range of biological problems: protein folding,[8] protein-ligand interactions,[9] and DNA base pairing.[10] Given the large number of G-quadruplex topologies possible,[1.2] SM-FS can potentially provide unique insights into their structure, function, and stability. Further, the recent development of theoretical analysis for SM approaches has enabled reliable estimates of the kinetic and thermodynamic parameters.[11] To the best of our knowledge, the AFM SM-FS has not been used to study G-quadruplex.[12] Herein, we present the first AFM based SM-FS study on a bi-molecular G-quadruplex system. We used a bi-molecular G-quadruplex formed between the AFM probe and a gold surface as the model system in this SM-FS study (see Fig. 1). The DNA sequences and their abbreviations are given in Table 1. To perform the SM-FS study, a gold-coated Si 3 N 4 probe and a flat gold
Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism
The Journal of Physical Chemistry B, 2021
G-quadruplexes play important roles in cellular regulatory functions, but despite significant experimental and theoretical efforts, their folding mechanisms remain poorly understood. In this context, we developed a T-jump experiment to access the thermal denaturation and renaturation dynamics of short intramolecular G-quadruplexes in vitro, on the time scale of a few hundred milliseconds. With this new setup , we compared the denaturation and renaturation kinetics of three antiparallel topologies made of the human telomeric sequences d[(5'-GGG(TTAGGG)3-3']/Na + and d[5'-AGGG(TTAGGG)3-3']/Na + and the thrombin-binding aptamer sequence d[5'-GGTTGGTGTGGTTGG-3']/K + , with those of the parallel topology made of the human CEB25 minisatellite d[5'-AAGGGTGGGTGTAAGTGTGGGTGGGT-3']/Na +. In all cases, exponential kinetics of the order of several hundred milliseconds were observed. Measurements performed for different initial temperatures revealed distinct denaturation and renaturation dynamics, ruling out a simple two-state mechanism. The parallel topology, in which all guanines adopt an anti conformation, displays much slower dynamics than antiparallel topologies associated with very low activation barriers. This behavior can be explained by the constrained conformational space due to the presence of the single-base propeller loops that likely hinders the movement of the coiled DNA strand and reduces the contribution of the entropy during the renaturation process at high temperatures.
Nucleic Acids Research, 2013
Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5 0 -(TTAGGG) 4 TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/ RNA polymerases with stall forces often <20 pN.
Energetic basis of AGCGA-rich DNA folding into a tetrahelical structure
Angewandte Chemie, 2019
It was recently discovered that, besides well-known G-quadruplexes and i-motifs, DNA may adopt another type of noncanonical structure called AGCGA-quadruplexes. Here, the folding of the VK2 fragment from the regulatory region of the PLEKHG3 gene is studied and, for the first time, the energetic contributions that stabilize this unique fold are described. Similarly to the B-DNA, it is stabilized by hydrophobic desolvation and, in contrast to G-quadruplexes, also by specific binding of water molecules. Compared to B-DNA, VK2 folding is enthalpically less favorable due to poorer basestacking interactions, resulting in substantial conformational flexibility. This entropically favorable conformational "breathing" stabilizes the AGCGA-quadruplexes. In conclusion, AGCGA-quadruplexes have a distinguishing thermodynamic fingerprint and the corresponding driving forces enabling their folding are consistent with the observed structural features.