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Ensemble Force Spectroscopy of a G‑Quadruplex Cluster on a Single-Molecule Platform
journal contributionposted on 2022-11-02, 18:05 authored by Pravin Pokhrel, Jiayi Wang, Sangeetha Selvam, Sagun Jonchhe, Shankar Mandal, Hanbin Mao
Single-molecule methods offer high sensitivities with precisions superior to bulk assays. However, these methods are low in throughput and cannot repetitively interrogate the same cluster of molecular units. In this work, we investigate a tandem array of G-quadruplexes on a single-molecule DNA template with a throughput of at least two orders of magnitude higher than single-molecule force spectroscopy. During mechanical unfolding by optical tweezers, the array of G-quadruplexes experiences identical force, temperature, and ionic conditions, which not only reduce environmental noise but also render unfolding transitions indistinguishable among individual G-quadruplexes. The resultant ensemble behaviors are analyzed by scanning force diagrams, which reveals accurate F1/2 values, where 50% of G-quadruplexes are unfolded. Independent of the number of G-quadruplexes (n > 15) contained in a cluster, F1/2 can effectively evaluate G-quadruplex ligands in a new method called differential scanning forcemetry. When the same G-quadruplex cluster is subject to a series of constant forces in force-jump experiments, unfolding rate constants of G-quadruplexes can be effectively evaluated as a function of force. The high precision demonstrated in all of these measurements reflects the power of repetitive sampling on the same cluster of single-molecule entities under identical conditions. Since biomolecules such as DNA, RNA, and proteins can be conveniently incorporated in a tandem array, we anticipate that this ensemble assay on single-molecule entities (EASE) provides a generic means of ensemble force spectroscopy to amalgamate the accuracy of ensemble measurements with the precision of single-molecule methods.
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