Molecular Nanomachines Research Articles

Thermal Energy Transport through Nonbonded Native Contacts in Protein.

Within the protein interior, where we observe various types of interactions, nonuniform flow of thermal energy occurs along the polypeptide chain and through nonbonded native contacts, leading to inhomogeneous transport efficiencies from one site to another. The folded native protein serves not merely as thermal transfer medium but, more importantly, as sophisticated molecular nanomachines in cells. Therefore, we are particularly interested in what sort of "communication" is mediated through native contacts in the folded proteins and how such features are quantitatively depicted in terms of local transport coefficients of heat currents. To address the issue, we introduced a concept of inter-residue thermal conductivity and characterized the nonuniform thermal transport properties of a small globular protein, HP36, using equilibrium molecular dynamics simulation and the Green-Kubo formula. We observed that the thermal transport of the protein was dominated by that along the polypeptide chain, while the local thermal conductivity of nonbonded native contacts decreased in the order of H-bonding > π-stacking > electrostatic > hydrophobic contacts. Furthermore, we applied machine learning techniques to analyze the molecular mechanism of protein thermal transport. As a result, the contact distance, variance in contact distance, and H-bonding occurrence probability during MD simulations are found to be the top three important determinants for predicting local thermal transport coefficients.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations.The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux.

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.

Pal power: Demonstration of the functional association of the Helicobacter pylori flagellar motor with peptidoglycan-associated lipoprotein (Pal) and its preliminary crystallographic analysis.

The bacterial flagellar motor is a molecular nanomachine, the assembly and regulation of which requires many accessory proteins. Their identity, structure and function are often discovered through characterisation of mutants with impaired motility. Here, we demonstrate the functional association of the Helicobacter pylori peptidoglycan-associated lipoprotein (HpPal) with the flagellar motor by analysing the motility phenotype of the ∆pal mutant, and present the results of the preliminary X-ray crystallographic analysis of its globular C-terminal domain HpPal-C. Purified HpPal-C behaved as a dimer in solution. Crystals of HpPal-C were grown by the hanging drop vapour diffusion method using medium molecular weight polyethylene glycol (PEG) Smear as the precipitating agent. The crystals belong to the primitive orthorhombic space group P1 with unit cell parameters a = 50.7, b = 63.0, c = 75.1 Å. X-ray diffraction data were collected to 1.8 Å resolution on the Australian Synchrotron beamline MX2. Calculation of the Matthews coefficient (VM=2.24 Å3/Da) and molecular replacement showed that the asymmetric unit contains two protein subunits. This study is an important step towards elucidation of the non-canonical role of H. pylori Pal in the regulation, or function of, the flagellar motor.

Depletion of SNORA33 Abolishes ψ of 28S-U4966 and Affects the Ribosome Translational Apparatus.

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33, but not SNORA22, reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.

Diversity and prevalence of type VI secretion system effectors in clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa is an opportunistic pathogen and a major driver of morbidity and mortality in people with Cystic Fibrosis (CF). The Type VI secretion system (T6SS) is a molecular nanomachine that translocates effectors across the bacterial membrane into target cells or the extracellular environment enabling intermicrobial interaction. P. aeruginosa encodes three T6SS clusters, the H1-, H2- and H3-T6SS, and numerous orphan islands. Genetic diversity of T6SS-associated effectors in P. aeruginosa has been noted in reference strains but has yet to be explored in clinical isolates. Here, we perform a comprehensive bioinformatic analysis of the pangenome and T6SS effector genes in 52 high-quality clinical P. aeruginosa genomes isolated from CF patients and housed in the Personalised Approach to P. aeruginosa strain repository. We confirm that the clinical CF isolate pangenome is open and principally made up of accessory and unique genes that may provide strain-specific advantages. We observed genetic variability in some effector/immunity encoding genes and show that several well-characterised vgrG and PAAR islands are absent from numerous isolates. Our analysis shows clear evidence of disruption to T6SS genomic loci through transposon, prophage, and mobile genetic element insertions. We identified an orphan vgrG island in P. aeruginosa strain PAK and five clinical isolates using in silico analysis which we denote vgrG7, predicting a gene within this cluster to encode a Tle2 lipase family effector. Close comparison of T6SS loci in clinical isolates compared to reference P. aeruginosa strain PAO1 revealed the presence of genes encoding eight new T6SS effectors with the following putative functions: cytidine deaminase, lipase, metallopeptidase, NADase, and pyocin. Finally, the prevalence of characterised and putative T6SS effectors were assessed in 532 publicly available P. aeruginosa genomes, which suggests the existence of accessory effectors. Our in silico study of the P. aeruginosa T6SS exposes a level of genetic diversity at T6SS genomic loci not seen to date within P. aeruginosa, particularly in CF isolates. As understanding the effector repertoire is key to identifying the targets of T6SSs and its efficacy, this comprehensive analysis provides a path for future experimental characterisation of these mediators of intermicrobial competition and host manipulation.

Unveiling the interdomain dynamics of type II DNA topoisomerase through all-atom simulations: Implications for understanding its catalytic cycle

Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed μs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

A programmable catalytic molecular nanomachine for highly sensitive protein and small molecule detection.

Herein, we report the construction of a programmable catalytic molecular nanomachine based on a cross-linked catalytic hairpin assembly (CCHA) reaction for the one-step highly sensitive detection of proteins and small molecules. In this system, when the recognition elements attached on split initiators bind to the target proteins, it can trigger the cascade of the CCHA reaction, resulting in the formation of many macromolecular fluorescent products for signaling. This platform couples the advantages of highly efficient DNA-based nanotechnology with specific protein-small molecule interactions. We demonstrated the sensitive detection of streptavidin and anti-digoxigenin antibody with detection limits as low as 48.8 pM and 0.85 nM, respectively. This nanomachine also demonstrated its flexibility in the nanomolar detection of corresponding small molecules, such as biotin and digoxigenin, using a competitive method. In addition, the nanomachine was robust enough to perform well with human serum samples. Overall, this programmable catalytic molecular nanomachine provides a versatile platform for the detection of proteins and small molecules by replacing the recognition elements, which can promote the development of DNA nanotechnology in disease diagnosis and therapeutic drug monitoring.

Multistep Transformations of DNA Origami Domino Array via Mechanical Forces

Nanostructures that display controllable motions and functions are indispensable to build molecular nanomachines. Structural DNA nanotechnology, enabling precise design of complex static and dynamic structures, can be used to construct molecular nanomachines. However, designing complex dynamic structures that present multiple motion behaviors with different stimuli‐responsiveness is full of challenges. Herein, multistep transformations of DNA origami domino array (DODA) triggered by mechanical forces are developed. The DODA consists of small, modular DNA units, which can be regulated by the external forces. Under the expanding and contraction forces exerted by the DNA trigger and G‐quadruplex (G4)‐functionalized loop strand, the units are reconfigured, leading to the programmable transformations of DODA among three conformations. This multistep transformation strategy is applied to construct chiral plasmonic metamolecules, and analyze K+ via fluorescence resonance energy transfer (FRET) analysis. Moreover, the strategy shows a diversified approach to build the DNA‐based nanodevices, and may find potential applications in various fields.

Secretome analysis of an environmental isolate Enterobacter sp. S-33 identifies proteins related to pathogenicity.

Enterobacter species are responsible for causing infections of the lower respiratory tract, urinary tract, meninges, etc. Proteins secreted by these species may act as determinants of host-pathogen interaction and play a role in virulence. Among the secreted proteins, the Type VI secretion system (T6SS) acts as a molecular nanomachine to deliver many effector proteins directly into prey cells in a contact-dependent manner. The secreted proteins may provide an idea for the interaction of bacteria to their environment and an understanding of the role of these proteins for their role in bacterial physiology and behaviour. Therefore, aim of this study was to characterize the secreted proteins in the culture supernatant by a T6SS bacterium Enterobacter sp. S-33 using nano-LC-MS/MS tool. Using a combined mass spectrometry and bioinformatics approach, we identified a total of 736 proteins in the secretome. Bioinformatics analysis predicting subcellular localization identified 110 of the secreted proteins possessed signal sequences. By gene ontology analysis, more than 80 proteins of the secretome were classified into biological or molecular functions. More than 20 percent of secretome proteins were virulence proteins including T6SS proteins, proteins involved in adherence and fimbriae formation, molecular chaperones, outer membrane proteins, serine proteases, antimicrobial, biofilm, exotoxins, etc. In summary, the results of the present study of the S-33 secretome provide a basis for understanding the possible pathogenic mechanisms and future investigation by detailed experimental approach will provide a confirmation of secreted virulence proteins in the exact role of virulence using the in vivo model.

Target-templated molecular nano-machine for auto-catalytic recycling amplification detection of low-abundant antibody in human serum

The development of convenient and sensitive sensing method capable of monitoring low-abundant antibody is appealing for pathogens diagnosis and vaccine immunotherapy evaluation. With regards to this, we herein constructed a simple molecular nano-machine for ultrasensitive monitoring of Anti-Digoxigenin (Anti-Dig) antibody via a target-initiated auto-cycling proximity recording (APR) amplification. One target bivalent Anti-Dig antibody can simultaneously recognize two Dig-labeled catalytic hairpin DNAs (Dig-HP1) and bring them into close distance to form the proximity catalytic probe pairs. Subsequently, the 3′-overhang of the FAM/Dabycl labeled hairpin signal probes hybridize on the exposure primer binding domain at Dig-HP1 template and initiate the APR reaction to cross open and extend Dig-HP1 to generate many elongated DNA duplexes with the help of Bst DNA polymerase. These DNA duplexes make the fluorophore and quencher away from each other and thereby produce dramatically enhanced fluorescence signal for sensitive detection of target Anti-Dig antibody down to 0.18 nM. Moreover, this method can selectively monitor Anti-Dig antibody against other interference proteins, and also be extended to detect Anti-Dig antibody in serum samples, confirming its potential for ultrasensitive detection of low-abundant antibodies for early disease therapy and vaccine evaluation.

Phylogenetic Classification and Functional Review of Autotransporters.

Autotransporters are the core component of a molecular nano-machine that delivers cargo proteins across the outer membrane of Gram-negative bacteria. Part of the type V secretion system, this large family of proteins play a central role in controlling bacterial interactions with their environment by promoting adhesion to surfaces, biofilm formation, host colonization and invasion as well as cytotoxicity and immunomodulation. As such, autotransporters are key facilitators of fitness and pathogenesis and enable co-operation or competition with other bacteria. Recent years have witnessed a dramatic increase in the number of autotransporter sequences reported and a steady rise in functional studies, which further link these proteins to multiple virulence phenotypes. In this review we provide an overview of our current knowledge on classical autotransporter proteins, the archetype of this protein superfamily. We also carry out a phylogenetic analysis of their functional domains and present a new classification system for this exquisitely diverse group of bacterial proteins. The sixteen phylogenetic divisions identified establish sensible relationships between well characterized autotransporters and inform structural and functional predictions of uncharacterized proteins, which may guide future research aimed at addressing multiple unanswered aspects in this group of therapeutically important bacterial factors.

Single nucleotide polymorphism determines constitutive versus inducible type VI secretion in Vibrio cholerae

Vibrio cholerae is a well-studied human pathogen that is also a common inhabitant of marine habitats. In both environments, the bacterium is subject to interbacterial competition. A molecular nanomachine that is often involved in such competitive behavior is the type VI secretion system (T6SS). Interestingly and in contrast to non-pandemic or environmental isolates, the T6SS of the O1 El Tor clade of V. cholerae, which is responsible for the ongoing 7th cholera pandemic, is largely silent under standard laboratory culture conditions. Instead, these strains induce their full T6SS capacity only under specific conditions such as growth on chitinous surfaces (signaled through TfoX and QstR) or when the cells encounter low intracellular c-di-GMP levels (TfoY-driven). In this study, we identified a single nucleotide polymorphism (SNP) within an intergenic region of the major T6SS gene cluster of V. cholerae that determines the T6SS status of the cell. We show that SNP conversion is sufficient to induce T6SS production in numerous pandemic strains, while the converse approach renders non-pandemic/environmental V. cholerae strains T6SS-silent. We further demonstrate that SNP-dependent T6SS production occurs independently of the known T6SS regulators TfoX, QstR, and TfoY. Finally, we identify a putative promoter region adjacent to the identified SNP that is required for all forms of T6SS regulation in V. cholerae.

Abstract 267: Light-activated molecular nanomachines kill bladder cancer cells

Abstract Introduction: Molecular nanomachines (MNMs) have been studied for their anti-tumor efficacy in a number of solid malignancies. They consist of small (1 nm) organic molecule-based motors that change conformation upon light activation and mechanically drill holes in the cell membrane resulting in cellular necrosis, representing a novel form of molecular mechanical therapy. Recently, we generated a blue light spectrum (395-405 nm)-activated MNMs which showed promising results in pancreatic cancer cell lines. Herein, we evaluate the in vitro efficacy in urothelial carcinoma, a disease characterized by high rates of recurrence, progression, and therapeutic resistance. Methods: MNMs activated by the blue light spectrum were synthesized. In vitro efficacy of the activated MNMs (8 µM in 0.1% DMSO) was investigated in TCCSUP and UC-3 human bladder cancer cell lines. Cells were incubated with MNMs for 30 minutes, then treatment groups were subjected to 300 mW/cm2 blue light (comparable to Blue Light Cystoscope) for 4 minutes. Control groups included MNMs without light, 0.1% DMSO (required for solubility of the MNMs) with light, and 0.1% DMSO without light. Cell viability was quantitatively evaluated utilizing propidium iodide and flow cytometry assay with each datapoint representing triplicates. Additionally, cells were grown and subjectively tested for cell adhesion to the culture dish at 1d following light and MNM treatment. In addition, we utilized the wound healing assay to detect changes in cell motility following MNM treatment. Results: TCCSUP cells exhibited almost 100% loss in viability when treated with light-activated MNMs. Solution only (0.1% DMSO) and unactivated MNMs exhibited less than 5% change in cell viability, whereas, solution and light exhibited 20% reduction in cell viability suggesting some light sensitivity of TCCSUP cells. Cell adhesion assay 1d post-treatment confirmed 100% cell death for the MNMs + light treated cells, while showing survival of cells with light only treatment. In addition, results from wound healing assay conducted on UC-3 cells demonstrated at 72 h, there was significant inhibition of cell motility in the treatment group (MNMs + light) compared to control groups. Conclusions: Light-activated MNM exhibit in vitro efficacy in multiple human bladder cancer cells lines. MNMs are a promising novel therapeutic modality for the intravesical treatment of bladder cancer, and there are no known resistance mechanisms. Further preclinical characterization of antitumor efficacy and tolerability are necessary in in vivo models. Citation Format: Supriya Nagaraju, Alexis van Venrooy, Patrick J. Hensley, Kelly K. Bree, Ciceron Ayala-Orozco, Nathan A. Brooks, David Izhaky, James M. Tour, Ashish M. Kamat. Light-activated molecular nanomachines kill bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 267.

Abstract 269: Harnessing the mechanical power of nanomachines to treat cancer: Light-activated molecular nanomachines kill melanoma and oral cancer cells

Abstract Introduction: Molecular nanomachines (MNMs) have been studied for their anti-tumor efficacy in a number of solid malignancies. They consist of small (1 nm) organic molecule-based motors that change conformation upon light activation and mechanically drill holes in the cell membrane resulting in cellular necrosis. That opens a novel way of molecular mechanical therapy. Recently, we generated a blue light spectrum (395-405 nm)-activated MNMs which showed promising results in pancreatic cancer cell lines. Herein, we evaluate the in vitro efficacy in various oral and skin cancers including squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma, a disease characterized by fast progression and therapeutic resistance. Methods: MNMs activated by the blue light spectrum were synthesized. In vitro efficacy of the activated MNMs (8 µM in 0.1% DMSO) was investigated in skin cancer cell lines. Cells were incubated with MNMs for 30 minutes, then treatment groups were subjected to 150 mW/cm2 blue light for 4 minutes or 300 mW/cm2 for 5 min. Control groups included MNMs without light, 0.1% DMSO (required for solubility of the MNMs) with light, and 0.1% DMSO without light. Cell viability was quantitatively evaluated utilizing propidium iodide (PI) and flow cytometry assay with each datapoint representing triplicates. Additionally, cells were grown and tested by clonogenic assay 1d and 9d following light and MNMs treatment. Results: B16-F10 (murine melanoma cancer cell line) treated with light-activated MNMs exhibited almost 100% loss in viability when treated with MNMs and light. Solution only (0.1% DMSO), unactivated MNMs and DMSO solution combined with light exhibited less than 5% change in cell viability suggesting that all the controls are non-cytotoxic evaluated by PI-flow cytometry at 3 h after treatment. Clonogenic assay 9 day post treatment confirmed 100% cell death for the MNMs + light treated cells, while showing nearly 80-90% survival of cells in the light only treatment control. Other treated cell lines include SCC cell lines (ROC 1 and ROC 3) showing 100% loss in cell viability when treated with MNMs and light. Conclusions: Light-activated MNM exhibit profound in vitro efficacy in multiple skin cancer cells lines. MNMs are a promising novel therapeutic modality for the treatment of oral and skin cancers, and there are no known resistance mechanisms for nanomechanical therapy. They are now being tested on murine tumor models of B16-F10 in C57BL/6 mice and further preclinical characterization of antitumor efficacy and tolerability are necessary in these studies. Citation Format: Ciceron Ayala-Orozco, Alexis van Venrooy, Dongdong Liu, Yewen Shi, Jacob Beckham, David Izhaky, James M. Tour, Jeffrey N. Myers, Roberto Rangel. Harnessing the mechanical power of nanomachines to treat cancer: Light-activated molecular nanomachines kill melanoma and oral cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 269.

Programmable Transformations of DNA Origami Made of Small Modular Dynamic Units.

Dynamic DNA origami has been employed for generating a rich repository of molecular nanomachines that are capable of sensing various cues and changing their conformations accordingly. The common design principle of the existing DNA origami nanomachines is that each dynamic DNA origami is programmed to transform in a specific manner, and the nanomachine needs to be redesigned to achieve a different form of transformation. However, it remains challenging to enable a multitude of controlled transformations in a single design of dynamic DNA nanomachine. Here we report a modular design method to programmatically tune the shapes of a DNA origami nanomachine. The DNA origami consists of small, modular DNA units, and the length of each unit can be selectively changed by toehold-mediated strand displacement. By use of different combinations of trigger DNA strands, modular DNA units can be selectively transformed, leading to the programmable reconfiguration of the overall dimensions and curvatures of DNA origami. The modular design of programmable shape transformation of DNA origami can find potential applications in more sophisticated molecular nanorobots and smart drug delivery nanocarriers.

Resolving cargo-motor-track interactions with bifocal parallax single-particle tracking

Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single-particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0–360°), polar angle, and three-dimensional (3D) displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only were well-known active intracellular transport and free diffusion observed, but new interactions (tight attachment and tethered rotation) were also discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.

Molecular Nanomachines Constructed from DNA Origami

1．はじめに