Using Thioflavin T as a biosensor for the detection of nucleic acid secondary structures

The purpose of this ongoing research project was to learn more about the compound Thioflavin T by developing methods using a UV/Vis spectrophotometer to study how this molecule interacts with other biomolecules. Thioflavin T (ThT), is a fluorescent dye that can bind proteins and nucleic acids and can be used to probe protein aggregation and unique nucleic acid secondary structures. It can be used as a biosensor to detect specific abnormal proteins called amyloid fibrils in biological samples. In this project, we will investigate how thioflavin T interacts with different nucleic acid secondary structures with the goal of developing a ThT-based biosensor for nucleic acid structures

Development of a Nucleic Acid Based Fluorescence ATP Biosensor

Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of biosensors for disease markers, environmental impact, and many other applications. In this research, we explored these unique properties of aptamers in developing a fluorescence-based biosensor for ATP (adenosine triphosphate) and related small molecules. The effectiveness of the biosensor was determined by measuring the binding affinity and specificity of the ATP biosensor on a molecular level, towards different, but structurally similar, ligands. We observed strong and similar binding affinity towards ATP and ATP analogs with Kd range (73-347 µM). However, when probed against other deoxyribonucleotide triphosphates (dNTPs), little to no binding was observed indicating the biosensor specifically targets only ATP analogs. The ATP aptamer sequence can also form noncanonical G4 secondary structure depending on the solution conditions. We investigated the involvement of the G-quartets in the aptamer sequence in ligand binding and found that both G-quartets contribute to ligand binding

Dual binding of an antibody and a small molecule increases the stability of TERRA G-quadruplex.

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.H.M. acknowledges support from NSF CHE-1026532. The Balasubramanian lab is supported by programme funding from Cancer Research UK.This is the final published version. It first appeared at http://onlinelibrary.wiley.com/doi/10.1002/anie.201408113/abstract;jsessionid=BB18FC03F2AF0C3EB95EC57CCBDB3DB9.f01t01

Single-Molecule Measurements of the Binding between Small Molecules and DNA Aptamers

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 (Δ<i><i>G</i></i>_unfold) between these two aptamers yields a Δ<i><i>G</i></i> of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (<i>K</i>_d) of 2.0 ± 0.2 μM, a value consistent with the <i>K</i>_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

G‑Quadruplex Structure in the ATP-Binding DNA Aptamer Strongly Modulates Ligand Binding Activity

Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040–0.042 μM for variants as compared to 0.15 μM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies

Direct Quantification of Loop Interaction and π–π Stacking for G‑Quadruplex Stability at the Submolecular Level

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