Prakash Shrestha

Single-molecule mechanochemical sensing using DNA origami nanostructures.

While single-molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single-molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7-tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet-derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single-molecule sensing with improved throughput.

Confined space facilitates G-quadruplex formation

Molecular simulations suggest that the stability of a folded macromolecule increases in a confined space due to entropic effects. However, due to the interactions between the confined molecular structure and the walls of the container, clear-cut experimental evidence for this prediction is lacking. Here, using DNA origami nanocages, we show the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. We induce targeted mechanical unfolding of the G-quadruplex while leaving the nanocage unperturbed. We find that the mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase with decreasing cage size. Compared to the case of diluted or molecularly crowded buffer solutions, the G-quadruplex inside the nanocage is significantly more stable, showing a 100 times faster folding rate. Our findings suggest the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside the polymerase machinery in cells.

Nascent RNA transcripts facilitate the formation of G-quadruplexes

Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls.

Yoctoliter Thermometry for Single‐Molecule Investigations: A Generic Bead‐on‐a‐Tip Temperature‐Control Module

A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.

A new concentration jump strategy reveals the lifetime of i-motif at physiological pH without force

Concentration jumps for kinetics measurement remain a challenge for single-molecule techniques, which have demonstrated superior signal-to-noise levels compared to ensemble average approaches. Currently, all concentration jumps use mixing strategies. Here, we report a simple and drastically different jump strategy by rapid transportation of molecules between two side-by-side laminar streams in 80 ms. This allowed us to measure the lifetime of bioactive DNA i-motif structures at physiological pH without force. We placed a human telomeric i-motif inside a DNA hairpin-based mechanical reporter. Since the folded or unfolded state of the hairpin correlates with that of the i-motif, by recording hairpin transitions, a half-life of ∼3 s was found for the DNA i-motif at neutral pH without force. Such a lifetime is sufficient for i-motif to interact with proteins to modulate cellular processes. We anticipate this concentration jump offers a generic platform to investigate single-molecule kinetics.

Mechanical properties of DNA origami nanoassemblies are determined by Holliday junction mechanophores

DNA nanoassemblies have demonstrated wide applications in various fields including nanomaterials, drug delivery and biosensing. In DNA origami, single-stranded DNA template is shaped into desired nanostructure by DNA staples that form Holliday junctions with the template. Limited by current methodologies, however, mechanical properties of DNA origami structures have not been adequately characterized, which hinders further applications of these materials. Using laser tweezers, here, we have described two mechanical properties of DNA nanoassemblies represented by DNA nanotubes, DNA nanopyramids and DNA nanotiles. First, mechanical stability of DNA origami structures is determined by the effective density of Holliday junctions along a particular stress direction. Second, mechanical isomerization observed between two conformations of DNA nanotubes at 10–35 pN has been ascribed to the collective actions of individual Holliday junctions, which are only possible in DNA origami with rotational symmetric arrangements of Holliday junctions, such as those in DNA nanotubes. Our results indicate that Holliday junctions control mechanical behaviors of DNA nanoassemblies. Therefore, they can be considered as ‘mechanophores’ that sustain mechanical properties of origami nanoassemblies. The mechanical properties observed here provide insights for designing better DNA nanostructures. In addition, the unprecedented mechanical isomerization process brings new strategies for the development of nano-sensors and actuators.

Mechanochemical Sensing: A Biomimetic Sensing Strategy.

Existing biosensors employ two major components: analyte recognition and signal transduction. Although specificity is achieved through analyte recognition, sensitivity is usually enhanced through a chemical amplification stage that couples the two main units in a sensor. Although highly sensitive, the extra chemical amplification stage complicates the sensing protocol. In addition, it separates the two elements spatiotemporally, reducing the real-time response of the biosensor. In this review, we discuss the new mechanochemical biosensors that employ mechanochemical coupling strategies to overcome these issues. By monitoring changes in the mechanical properties of a single-molecule template upon analyte binding, single-molecule sensitivity is reached. As chemical amplification becomes unnecessary in this single-molecule mechanochemical sensing (SMMS) strategy, real-time sensing is achieved.

Decreased water activity in nanoconfinement contributes to the folding of G-quadruplex and i-motif structures

Significance Nanocavities in cellular compartments or molecular assemblies are commonplace in nature. Nanoconfinement has demonstrated significant effects on the folding of biomolecules, which is mediated by hydration waters. Therefore, understanding the property of water molecules in nanocavities is instrumental to elucidate the confinement effect. By exploiting the known numbers of waters lost during the folding of DNA G-quadruplex and i-motif structures, we quantified that water activities decreased within smaller DNA origami nanocages. This effect is more pronounced than that induced by cosolutes such as polyethylene glycol (PEG). In addition, loss of water molecules during the folding of G-quadruplex and i-motif governs the folding of biomolecules in nanoconfinement. Due to the small size of a nanoconfinement, the property of water contained inside is rather challenging to probe. Herein, we measured the amount of water molecules released during the folding of individual G-quadruplex and i-motif structures, from which water activities are estimated in the DNA nanocages prepared by 5 × 5 to 7 × 7 helix bundles (cross-sections, 9 × 9 to 15 × 15 nm). We found water activities decrease with reducing cage size. In the 9 × 9-nm cage, water activity was reduced beyond the reach of regular cosolutes such as polyethylene glycol (PEG). With this set of nanocages, we were able to retrieve the change in water molecules throughout the folding trajectory of G-quadruplex or i-motif. We found that water molecules absorbed from the unfolded to the transition states are much fewer than those lost from the transition to the folded states. The overall loss of water therefore drives the folding of G-quadruplex or i-motif in nanocages with reduced water activities.

Single-Molecule Mechanochemical pH Sensing Revealing the Proximity Effect of Hydroniums Generated by an Alkaline Phosphatase

Due to the fast diffusion, small molecules such as hydronium ions (H3O+) are expected to be homogeneously distributed, even close to the site-of-origin. Given the importance of H3O+ in numerous processes, it is surprising that H3O+ concentration ([H3O+]) has yet to be profiled near its generation site with nanometer resolution. Here, we innovated a single-molecule method to probe [H3O+] in nanometer proximity of individual alkaline phosphatases. We designed a mechanophore with cytosine (C)-C mismatch pairs in a DNA hairpin. Binding of H3O+ to these C-C pairs changes mechanical properties, such as stability and transition distance, of the mechanophore. These changes are recorded in optical tweezers and analyzed in a multivariate fashion to reduce the stochastic noise of individual mechanophores. With this method, we found [H3O+] increases in the nanometer vicinity of an active alkaline phosphatase, which supports that the proximity effect is the cause for increased rates in cascade enzymatic reactions.