Chiran Ghimire

Mutually Exclusive Formation of G-Quadruplex and i-Motif Is a General Phenomenon Governed by Steric Hindrance in Duplex DNA.

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to possible offset arrangements between G-quadruplex and i-motif in opposite strands. The mutual exclusivity revealed here sets a molecular basis for cells to efficiently coordinate opposite biological activities of G-quadruplex and i-motif at the same dsDNA location.

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.

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.

Controlled Particle Collision Leads to Direct Observation of Docking and Fusion of Lipid Droplets in an Optical Trap

As an intracellular organelle, phospholipid-coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusion. Unlike phospholipid vesicles that are well-known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffer at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and physical fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 μs after postsynchronization analysis. Our method revealed that the rate-limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the physical fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ∼46 ms lifetime in POPC-coated lipid droplets, but not in bare triolein droplets.

A Molecular Tuning Fork in Single-Molecule Mechanochemical Sensing†

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real-time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single-molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence). Reminiscent of a tuning fork that vibrates at a fixed frequency, the device was classified as a molecular tuning fork (MTF). By monitoring the lifetime of the folded and unfolded hairpins with equal populations, we were able to differentiate between the mono- and bivalent binding modes during individual antibody-antigen binding events. We anticipate these mechanospectroscopic concepts and methods will be instrumental for the development of novel bioanalyses.

Dissecting Cooperative Communications in a Protein with a High‐Throughput Single‐Molecule Scalpel

Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double-stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all-or-none and partially cooperative events, which represent strong and weak high-order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all-or-none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all-or-none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.