Dynamic Structures and Fast Transition Kinetics of Oxidized G-Quadruplexes.

8-oxoguanines (8-oxoG) in cells form compromised G-quadruplexes (GQs), which may vary GQ mediated gene regulations. By mimicking molecularly crowded cellular environment using 40% DMSO or sucrose, here it is found that oxidized human telomeric GQs have stabilities close to the wild-type (WT) GQs. Surprisingly, while WT GQs show negative formation cooperativity between a Pt(II) binder and molecularly crowded environment, positive cooperativity is observed for oxidized GQ formation. Single-molecule mechanical unfolding reveals that 8-oxoG sequence formed more diverse and flexible structures with faster folding/unfolding transition kinetics, which facilitates the Pt(II) ligand to bind the best-fit structures with positive cooperativity. These findings offer new understanding on structures and properties of oxidized G-rich species in crowded environments. They also provide insights into the design of better ligands to target oxidized G-rich structures formed under oxidative cell stress.

Zeptoliter DNA Origami Reactor to Reveal Cosolute Effects on Nanoconfined G-Quadruplexes.

Cellular environments such as nanoconfinement and molecular crowding can change biomolecular properties. However, in nanoconfinement, it is extremely challenging to investigate effects of crowding cosolutes on macromolecules. By using optical tweezers, here, we elucidated the effects of hexaethylene glycol (HEG) on the mechanical stability of a telomeric G-quadruplex (GQ) in a zeptoliter DNA origami reactor (zepto-reactor). When HEG molecules were introduced in the GQ-containing zepto-reactor at different positions, we found that the GQ species split into two equilibrated populations, reflecting diverse effects of the oligoethylene glycol on the GQ via either a long-range dehydration effect or direct interactions. When the number of HEG molecules was increased, the stability of the GQ unexpectedly decreased, suggesting that the direct destabilizing interaction between the GQ and HEG is dominating over the long-range stabilizing dehydration effects of the HEG in hydrophilic nanocavities. These findings indicate that a nanoconfined environment can alter regular effects of cosolutes on biomacromolecules.

Single-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex.

Binding between a ligand and a receptor is a fundamental step in many natural or synthetic processes. In biosensing, a tight binding with a small dissociation constant (Kd) between the probe and analyte can lead to superior specificity and sensitivity. Owing to their capability of evaluating competitors, displacement assays have been used to estimate Kd at the ensemble average level. At the more sensitive single-molecule level, displacement assays are yet to be established. Here, we developed a single-molecule displacement assay (smDA) in an optical tweezers instrument and used this innovation to evaluate the binding of the L2H2-6OTD ligands to human telomeric DNA G-quadruplexes. After measuring Kd of linear and dendrimer L2H2-6OTD ligands, we found that dendrimer ligands have enhanced binding affinity to the G-quadruplexes due to their polyvalent geometry. This increased binding affinity enhanced inhibition of telomerase elongation on a telomere template in a Telomerase Repeated Amplification Protocol (TRAP). Our experiments demonstrate that the smDA approach can efficiently evaluate binding processes in chemical and biological processes.

Chirality transmission in macromolecular domains.

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.

Dissection of nanoconfinement and proximity effects on the binding events in DNA origami nanocavity.

Both ligand binding and nanocavity can increase the stability of a biomolecular structure. Using mechanical unfolding in optical tweezers, here we found that a DNA origami nanobowl drastically increased the stability of a human telomeric G-quadruplex bound with a pyridostatin (PDS) ligand. Such a stability change is equivalent to >4 orders of magnitude increase (upper limit) in binding affinity (Kd: 490 nM → 10 pM (lower limit)). Since confined space can assist the binding through a proximity effect between the ligand-receptor pair and a nanoconfinement effect that is mediated by water molecules, we named such a binding as mechanochemical binding. After minimizing the proximity effect by using PDS that can enter or leave the DNA nanobowl freely, we attributed the increased affinity to the nanoconfinement effect (22%) and the proximity effect (78%). This represents the first quantification to dissect the effects of proximity and nanoconfinement on binding events in nanocavities. We anticipate these DNA nanoassemblies can deliver both chemical (i.e. ligand) and mechanical (i.e. nanocavity) milieus to facilitate robust mechanochemical binding in various biological systems.

Direct Measurement of Intermolecular Mechanical Force for Nonspecific Interactions between Small Molecules.

Mechanical force can evaluate intramolecular interactions in macromolecules. Because of the rapid motion of small molecules, it is extremely challenging to measure mechanical forces of nonspecific intermolecular interactions. Here, we used optical tweezers to directly examine the intermolecular mechanical force (IMMF) of nonspecific interactions between two cholesterols. We found that IMMFs of dimeric cholesterol complexes were dependent on the orientation of the interaction. The surprisingly high IMMF in cholesterol dimers (∼30 pN) is comparable to the mechanical stability of DNA secondary structures. Using Hess-like cycles, we quantified that changes in free energy of solubilizing cholesterol (ΔGsolubility) by ß-cyclodextrin (ßCD) and methylated ßCD (Me-ßCD) were as low as -16 and -27 kcal/mol, respectively. Compared to the ΔGsolubility of cholesterols in water (5.1 kcal/mol), these values indicated that cyclodextrins can easily solubilize cholesterols. Our results demonstrated that the IMMF can serve as a generic and multipurpose variable to dissect nonspecific intermolecular interactions among small molecules into orientational components.

Measurement of Single-Molecule Forces in Cholesterol and Cyclodextrin Host-Guest Complexes.

Biological host molecules such as ß-cyclodextrins (ß-CDs) have been used to remove cholesterol guests from membranes and artery plaques. In this work, we calibrated the host-guest intermolecular mechanical forces (IMMFs) between cholesterol and cyclodextrin complexes by combining single-molecule force spectroscopy in optical tweezers and computational molecular simulations for the first time. Compared to native ß-CD, methylated beta cyclodextrins complexed with cholesterols demonstrated higher mechanical stabilities due to the loss of more high-energy water molecules inside the methylated ß-CD cavities. This result is consistent with the finding that methylated ß-CD is more potent at solubilizing cholesterols than ß-CD, suggesting that the IMMF can serve as a novel indicator to evaluate the solubility of small molecules such as cholesterols. Importantly, we found that the force spectroscopy measured in such biological host-guest complexes is direction-dependent: pulling from the alkyl end of the cholesterol molecule resulted in a larger IMMF than that from the hydroxyl end of the cholesterol molecule. Molecular dynamics coupled with umbrella sampling simulations further revealed that cholesterol molecules tend to enter or leave from the wide opening of cyclodextrins. Such an orientation rationalizes that cyclodextrins are rather efficient at extracting cholesterols from the phospholipid bilayer in which hydroxyl groups of cholesterols are readily exposed to the hydrophobic cavities of cyclodextrins. We anticipate that the IMMF measured by both experimental and computational force spectroscopy measurements help elucidate solubility mechanisms not only for cholesterols in different environments but also to host-guest systems in general, which have been widely exploited for their solubilization properties in drug delivery, for example.

Mechanochemical properties of DNA origami nanosprings revealed by force jumps in optical tweezers.

By incorporating pH responsive i-motif elements, we have constructed DNA origami nanosprings that respond to pH changes in the environment. Using an innovative force jump approach in optical tweezers, we have directly measured the spring constants and dynamic recoiling responses of the DNA nanosprings under different forces. These DNA nanosprings exhibited 3 times slower recoiling rates compared to duplex DNA backbones. In addition, we observed two distinct force regions which show different spring constants. In the entropic region below 2 pN, a spring constant of ∼0.03 pN nm-1 was obtained, whereas in the enthalpic region above 2 pN, the nanospring was 17 times stronger (0.5 pN nm-1). The force jump gave a more accurate measurement on nanospring constants compared to regular force ramping approaches, which only yielded an average spring constant in a specific force range. Compared to the reported DNA origami nanosprings with a completely different design, our nanospring is up to 50 times stiffer. The drastic increase in the spring constant and the pH responsive feature allow more robust applications of these nanosprings in many mechanobiological processes.

Plastome of Saraca asoca (Detarioideae, Fabaceae): Annotation, comparison among subfamily and molecular typing.

Saraca asoca (Roxb.) Willd. (subfamily Detarioideae, family Fabaceae) is a perennial evergreen sacred medicinal tree classified under 'vulnerable' by the IUCN. The chloroplast (cp) genome/plastome which follows uniparental inheritance contains many useful genetic information because of its conservative rate of evolution. The assembled cp genome of S. asoca which maps as a conserved circular structure revealed extensive rearrangement in gene organization, comprising total length 160,003 bp including LSC, SSC, IRa, and IRb, and GC content was 35.26%. Herein a set of rbcL and matK gene were established using molecular phylogenetic analyses for molecular typing of S. asoca.

Cooperative Heteroligand Interaction with G-Quadruplexes Shows Evidence of Allosteric Binding.

Although allosteric binding of small molecules is commonplace in protein structures, it is rather rare in DNA species such as G-quadruplexes. By using CD melting, here, we found binding of the small-molecule ligands PDS and L2H2-6OTD to the telomeric DNA G-quadruplex was cooperative. Mass spectrometry indicated a 1:1:1 ratio in the ternary binding complex of the telomeric G-quadruplex, PDS, and L2H2-6OTD. Compared to the binding of each individual ligand to the G-quadruplex, single-molecule mechanical unfolding assays revealed a significantly decreased dissociation constant when one ligand is evaluated in the presence of another. This demonstrates that cooperative binding of PDS and L2H2-6OTD to the G-quadruplex is allosteric, which is also supported by the mass spectra data that indicated the ejection of coordinated sodium ions upon binding of the heteroligands to the G-quadruplex. The unprecedented observation of the allosteric ligand binding to higher-ordered structures of DNA may help to design more effective ligands to target non-B DNA species involved in many critical cellular processes.

Duplex DNA Is Weakened in Nanoconfinement.

For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, the properties of the physiologically prevalent B-DNA duplex have not been elucidated inside the nanocavity. Using a 17-bp DNA duplex in the form of a hairpin stem, here, we probed folding and unfolding transitions of the hairpin DNA duplex inside a DNA origami nanocavity. Compared to the free solution, the DNA hairpin inside the nanocage with a 15 × 15 nm cross section showed a drastic decrease in mechanical (20 → 9 pN) and thermodynamic (25 → 6 kcal/mol) stabilities. Free energy profiles revealed that the activation energy of unzipping the hairpin DNA duplex decreased dramatically (28 → 8 kcal/mol), whereas the transition state moved closer to the unfolded state inside the nanocage. All of these indicate that nanoconfinement weakens the stability of the hairpin DNA duplex to an unexpected extent. In a DNA hairpin made of a stem that contains complementary telomeric G-quadruplex (GQ) and i-motif (iM) forming sequences, formation of the Hoogsteen base pairs underlining the GQ or iM is preferred over the Watson-Crick base pairs in the DNA hairpin. These results shed light on the behavior of DNA in nanochannels, nanopores, or nanopockets of various natural or synthetic machineries. It also elucidates an alternative pathway to populate noncanonical DNA over B-DNA in the cellular environment where the nanocavity is abundant.

Chaperone-Assisted Host-Guest Interactions Revealed by Single-Molecule Force Spectroscopy.

The recent discovery of ultra-high binding affinities in cucurbit[7]uril (CB7)-based host-guest pairs in an aqueous environment has rendered CB7 a rather attractive material in analytical and biomedical applications. Due to the lack of a molecular platform that can follow the same host-guest complex during repetitive mechanical measurements, however, mechanical stabilities of the CB7 system have not been revealed, hindering its potential to rival widely used conjugation pairs, such as streptavidin-biotin. Here, we assembled a DNA template in which a flexible DNA linker was exploited to keep the host (CB7) and guest (adamantane) in proximity. This platform not only increased the efficiency of the single-molecule characterization in optical tweezers but also clearly revealed mechanical features of the same host-guest complex. We found that positively charged adamantane guest demonstrated higher mechanical stability (49 pN) than neutral adamantane (44 pN), a trend consistent with the chemical affinity between guest molecules and the CB7 host. Surprisingly, we found that a hexyl group adjacent to the adamantane served as a chaperone to assist the formation of the adamantane-CB7 pairs. The discovery of an unprecedented chaperone-assisted interaction mechanism provides new approaches to efficiently assemble host-guest-based supramolecules with increased mechanical stabilities, which can be exploited in various biomedical, biosensing, and materials fields.

Single-Molecule Topochemical Analyses for Large-Scale Multiplexing Tasks.

Multitasking is the pivotal feature in next-generation chemo- or bioanalyses. However, simultaneous analyses rarely exceed over three different tasks, which is ascribed to the limited space to accommodate analyzing units and the compromised signal-to-noise (S/N) level as the number of tasks increases. Here, by leveraging superior S/N of single-molecule techniques, we analyzed five microRNA biomarkers by spatially encoding miRNA recognition units with nanometers resolution in a DNA template, while decoding the analyte binding temporally in seconds. The hairpin stem is interspersed by internal loops to encode recognition units for miRNA. By mechanical unfolding of the hairpin, individual internal loops are sequentially interrogated for the binding of each miRNA. Using this so-called topochemical spatiotemporal analysis, we were able to achieve subpicomolar detection limits of miRNAs. We anticipate that this new single-molecule topochemical analysis can massively analyze single-molecule targets.

Submolecular dissection reveals strong and specific binding of polyamide-pyridostatin conjugates to human telomere interface.

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA-ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant (Kd) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found Kd of 0.8 nM for PA-PDS, which is much lower than PDS (Kd ∼ 450 nM) or PA (Kd ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.

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

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.