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′-(TTAGGG)4TTA, 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.
The 3' human telomeric overhang provides ample opportunities for the formation and interaction of G-quadruplexes, which have shown impacts on many biological functions including telomerase activities in the telomere region. However, in the few investigations on DNA constructs that approach to the full length of the human telomeric overhang, the presence of higher-order quadruplex-quadruplex interactions is still a subject of debate. Herein, we employed dynamic splint ligation (DSL) to prepare a DNA construct, 5'-(TTAGGG)24 or 24G, which has the length comparable to the full stretch of 3' human telomeric overhang. Using mechanical unfolding assays in laser tweezers, we observed a minor population (∼5%) of higher-order interactions between G-quadruplexes, while the majority of the quadruplexes follow the bead-on-a-string model. Analyses on the noninteracting G-quadruplexes in the 24G construct showed features similar to those of the stand-alone G-quadruplexes in the 5'-(TTAGGG)4 (4G) construct. As each 24G construct contains as many as six G-quadruplexes, this method offers increased throughput for the time-consuming mechanical unfolding experiments of non-B DNA structures.
Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer-ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5'-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (ΔG(unfold)) between these two aptamers yields a ΔG of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K(d)) of 2.0 ± 0.2 μM, a value consistent with the K(d) obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.
Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120–180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.
Potential functions: By following the unfolding and refolding of individual human RNA telomeric (TERRA) G-quadruplexes (GQs) in laser tweezers, the mechanical stability and transition kinetics of RNA GQs are obtained. Comparison between TERRA and DNA GQs suggests their different regulatory capacities for processes associated with human telomeres.
Single-stranded DNA (ssDNA) and RNA regions that include at least four closely spaced runs of three or more consecutive guanosines strongly tend to fold into stable G-quadruplexes (G4s). G4s play key roles as DNA regulatory sites and as kinetic traps that can inhibit biological processes, but how G4s are regulated in cells remains largely unknown. Here, we developed a kinetic framework for G4 disruption by DEAH-box helicase 36 (DHX36), the dominant G4 resolvase in human cells. Using tetramolecular DNA and RNA G4s with four to six G-quartets, we found that DHX36-mediated disruption is highly efficient, with rates that depend on G4 length under saturating conditions (k cat ) but not under subsaturating conditions (k cat /K M ). These results suggest that a step during G4 disruption limits the k cat value and that DHX36 binding limits k cat /K M . Similar results were obtained for unimolecular DNA G4s. DHX36 activity depended on a 3′ ssDNA extension and was blocked by a polyethylene glycol linker, indicating that DHX36 loads onto the extension and translocates 3′-5′ toward the G4. DHX36 unwound dsDNA poorly compared with G4s of comparable intrinsic lifetime. Interestingly, we observed that DHX36 has striking 3′-extension sequence preferences that differ for G4 disruption and dsDNA unwinding, most likely arising from differences in the rate-limiting step for the two activities. Our results indicate that DHX36 disrupts G4s with a conventional helicase mechanism that is tuned for great efficiency and specificity for G4s. The dependence of DHX36 on the 3′-extension sequence suggests that the extent of formation of genomic G4s may not track directly with G4 stability. ___________________________________Single-stranded DNA and RNA segments that include at least four closely-spaced runs of three or more consecutive guanosine nucleotides have a strong intrinsic propensity to fold into stable G-quadruplex structures (G4s) (1) (Fig. 1A, top left). The genomes of humans and many other eukaryotes are replete with putative G4-forming sequences, and pronounced, conserved patterns have been observed in their distribution, suggesting that Gquadruplex structures form at least transiently in cells and perform conserved functions (2-6). Supporting this hypothesis, G4s have been detected in cells for both .To function as regulatory elements, G4s must be folded and disrupted in a regulated way. In addition, processes that require single-stranded DNA or RNA, such as replication and translation, require that G4s be temporarily disrupted. The high stability and long intrinsic lifetime of G4s suggest Mechanism of G4 disruption by DHX362 that their efficient disruption requires the activity of ATP-dependent enzymes such as helicases.Supporting this view, incorporation of sequences predicted to form particularly stable G4s leads to genetic instability in yeast, suggesting that these G4 structures are not processed efficiently and pose blocks to replication (12). Indeed, several helicases have been shown to possess G4 disruption acti...
The well-demonstrated biological functions of DNA G-quadruplex inside cells call for small molecules that can modulate these activities by interacting with G-quadruplexes. However, the paucity of the understanding of the G-quadruplex stability contributed from submolecular elements, such as loops and tetraguanine (G) planes (or G-quartets), has hindered the development of small-molecule binders. Assisted by click chemistry, herein, we attached pulling handles via two modified guanines in each of the three G-quartets in human telomeric G-quadruplex. Mechanical unfolding using these handles revealed that the loop interaction contributed more to the G-quadruplex stability than the stacking of G-quartets. This result was further confirmed by the binding of stacking ligands, such as telomestatin derivatives, which led to similar mechanical stability for all three G-quartets by significant reduction of loop interactions for the top and bottom G-quartets. The direct comparison of loop interaction and G-quartet stacking in G-quadruplex provides unprecedented insights for the design of more efficient G-quadruplex-interacting molecules. Compared to traditional experiments, in which mutations are employed to elucidate the roles of specific residues in a biological molecule, our submolecular dissection offers a complementary approach to evaluate individual domains inside a molecule with fewer disturbances to the native structure.
In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ–ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.
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