Biomolecular machines lie at the heart of all cellular functioning. As understanding of these molecular machines has increased, researchers have made significant progress in designing and constructing synthetic versions using a wide variety of materials as building blocks. As the sophistication of artificial molecular machines increases, their potential applications expand. In the biopharmaceutical industry, novel molecular machines are being investigated for controlled and targeted drug delivery, as well as a host of other uses.

Many Natural Biological Molecular Machines

Biological functioning can be viewed as driven by cellular machinery comprising several types of molecules, including nucleic acids and proteins. By working together, these molecules form natural biomolecular machines that respond in various ways to different types of stimuli, including the exertion of forces or the creation of motion.¹ These activities enable key intracellular and intercellular functions and information transfer.²

Over 400 protein-based molecular machines have been identified to date.³ They include DNA helicases and chromatin remodelers, which regulate the maintenance and expression of genetic data; RNA helicases, which are involved in detecting viruses and recognizing molecular patterns; and transporters that facilitate the intracellular and transmembrane transport of biomolecular substances.³ Other specific examples of natural biomolecular machines include the replisome, which replicates DNA in dividing cells; transcription machinery for copying genetic information; the ribosome, which translates the genetic code in RNA into proteins; the spliceosome, which cuts and reforms RNA, enabling the translation of multiple proteins from one RNA; dynein, which delivers proteins to the right locations; and ATP synthases, which produce adenosine triphosphate, the main form of energy that drives cellular processes.²

Given that natural biomolecular machines are essential to most cellular functions, it should be no surprise that deficiencies or mutations of the genes encoding their component proteins typically result in some form of disease.² That may include common illnesses such a cancer, diabetes, and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. As understanding of these biomolecular machines increases, some researchers are seeking to identify opportunities to target malfunctioning components to treat or cure diseases, while others are seeking to design artificial molecular machines that can be used not only for therapeutics –– largely for drug delivery –– but used in diagnostic applications and for fundamental research and biomedical engineering.¹

These synthetic molecular machines are molecular structures that can rotate unidirectionally in a controlled manner in response to stimuli, resulting in a mechanical action.”⁴ Ultraviolet (UV) light has been a widely used stimulus in early research efforts, as it is noninvasive and relatively easy to control, but other options are needed for clinical applications because UV radiation damages cells. Chemical stimuli are thus also being investigated.

Failed Molecular Machines as Drug Targets

Access to a growing body of genetic information has led to many advances in medicine in recent years. In the realm of molecule machines, there have been many discoveries regarding the interconnection of failed molecular machine functioning and cancer, cystic fibrosis, and other diseases. Point mutations in the genes encoding molecular machine proteins often lead to rare genetic disorders. Tumor mutations leverage molecular machines for cancer growth. The cystic fibrosis transporter, when malfunctioning, causes the disease.

At this stage, only a few therapeutics have been developed to repair and/or restore failed natural biomolecular machines.³ Drugs are on the market today that modulate the cystic fibrosis transporter. Prilosec (omeprazole) treats heartburn, damaged esophagi, stomach ulcers, and gastroesophageal reflux disease by targeting the H⁺/K⁺ ATPase, and etoposide targets the DNA topoisomerase II machinery, which is critical to DNA replication and cell division. However, most approved drugs targeting molecular machines were discovered serendipitously.

MOMA Therapeutics is one company looking to use a systematic approach to discovering therapeutics that target failed biomolecular machines.³ Its efforts are focused on exploiting the reliance of molecular machines on highly specific changes in protein conformation. The company is evaluating the conformational states of natural molecular machines to identify small molecules that can potentially bind to them and affect their function. The company will apply its systematic approach to different sub-families of natural molecular machines with the goal of discovering and developing novel medicines for patients in need.

Many Types of Bioactive Synthetic Molecular Machines

While natural biomolecular machines comprise nucleic acids (and thus DNA and RNA sequences) and proteins, researchers have not limited themselves to these natural building blocks with constructing synthetic molecular machines. In addition to proteins, RNA, and DNA, organic molecules and various types of nanoparticles have been leverages to construct artificial molecular machines.¹ The 2016 Nobel Prize in Chemistry was awarded to scientists who created molecular machines based on organic molecules that use chemical fuels, light, and redox reactions to drive molecular machines with unidirectional motion that are able to perform complex tasks.⁵ Since then, much work has focused on DNA-based molecular machines using complex DNA origami to generate two- and three-dimensional structures with wide-ranging functional capabilities.¹

Molecular machines were originally developed in solution, but much work in recent years has focused on creating synthetic versions anchored on various surfaces. In solution, a systems chemistry approach is required to create complex mixtures that can act as molecular machines and affect their environment. Different components of such mixtures must interact with one another to control chemical reactivities and structural assembly, which is quite challenging. Attaching components to a nanoscale gold surface allows for better control.⁶ Both light and chemical inputs have been used as fuel.

Crystals have been used to design molecular machines, generally by imparting stress that causes bending, resulting in performance as an actuator.⁶ That requires rapid (picosecond) switching in the solid state through some type of isomerization or other reversible reaction, often with photoactivation. Diarylethenes (DAs) have been used for this type of synthetic molecular machine.

Crystals are brittle and of limited size, which has led to interest in other more flexible types of materials, such as metal–organic frameworks (MOFs).⁶ MOFs also have a much greater inherent free volume for switching in the solid state but require careful crystal engineering to allow such changes to occur. In addition, larger motions can be achieved when using rotaxanes in the framework.

Surface-bound artificial molecular machines largely leverage nanoparticle technologies. Attaching machines to surfaces imparts order and provides a means of connecting machines together so they can have macroscopic impacts.⁶ Surface-bound rotaxanes, for instance, impart stress on the surface by moving and bend in return. Azobenzene moieties attached to small peptides can inhibit the activity of kinesin when in the trans phase, as part of the H⁺/K⁺ ATPase molecular machine. Of particular interest is the high level of control possible; using a specific activation wavelength, it is possible to stop the movement of just one of many microtubules in the H⁺/K⁺ ATPase machine. Rotaxanes have also been self-assembled on gold cantilevers using macrocyclic compounds, with movement of the cantilever driven by a redox reaction. Such synthetic molecular machines can move cargoes on surfaces and potentially be used in the construction of nanoelectromechanical, lab-on-a-chip, or microfluidic devices.

Molecular machines formed using liquid crystal (LC) polymers/elastomers have been widely studied and show promise for future applications.⁶ The polymers retain their order and allow for structural changes on the microscopic scale because tiny movements are amplified via long-range self-assembly. Polyazobenzenes are commonly employed as photo switches that undergo a trans–cis isomerization to deform the liquid crystal. Others have been derived from hydrazones, and there are examples of chemically and electrochemically activated LCs.

Amorphous polymeric systems have been less studied as molecular machines because of their inherently non-organized structure, which makes ordered motion difficult.⁶ Some researchers have begun to find ways to leverage collective molecular motion to overcome this challenge. Attaching such systems to surfaces helps to impart directionality.

The above examples all depend on light activation, but artificial molecular machines have been developed that are chemically activated.⁶ Two challenges to this approach are overcoming reversibility and managing by-products (waste) from the chemical reactions. Steric barriers can be leveraged to create directional motion. For instance, an artificial molecular machine has been devised using a small benzylic amide macrocycle in a [2]catenane to control the rate of an acylation reaction to attach 9-fluorenylmethoxycarbonyl groups. Rotaxanes have also been used to create various artificial molecular machines, including a pump. These systems operate in solution.

Another example specifically targeting drug-delivery applications is the functionalization of mesoporous nanoparticles with artificial molecular machines.⁷ Mesoporous nanoparticle have been extensively studied for drug delivery owing to their stability, ability to encapsulate a wide variety of drug substances (tunability), and functionalizability, which enables targeting of specific cells/tissues. Researchers are combining these advantages with the benefits of synthetic molecular machines based on rotaxanes, pseudorotaxanes, and azobenzenes to achieve controlled release of highly targeted therapeutics in response to various stimuli, including chemical reduction/oxidation, light, magnetic fields, enzymes, and pH changes.

Evolution of Synthetic DNA-Based Molecular Machines

Some of the greatest achievements with synthetic molecular machines have been realized using DNA as the building blocks, the first examples of which appeared in the 1980s.¹ Double-stranded DNA (dsDNA) is attractive as a material for constructing molecular machines because certain helix structures of known dimensions can be generated using specific complementary sequences and be “programmed” using hybridization. While the first DNA molecular machines were two-dimensional and formed using simple sequences, highly complex, three-dimensional architectures have since been developed using multiple types of DNA origami.

DNA is attractive as a building block for artificial molecular machines because it is flexible, allowing a wide range of structures, and because it can be programmed, allowing for formation of structures and imparting of behaviors in a controlled manner.¹ Tetrahedrons, cubes, smiley faces, monoliths with pleated layers, ellipsoids, dodecahedrons, and many other shapes have been generated. As with organic-based artificial molecular machines, work with DNA has also transitioned from solution to machines supported on surfaces to provide directionality, cooperative operation, and greater functionality.

A new form of DNA referred to as single-stranded tile (SST) DNA that contains four concatenated sequences of nearly identical length has been used to create tubule-shaped DNA-based molecular machaines.¹ The DNA “brick,” meanwhile, is a 32-nucleotide-long single-stranded DNA (ssDNA) with a square lattice arrangement of parallel helices that can be used to form not only two- and three-dimensional shapes but also crystal lattices with channels and pores.

“Scaffolded DNA origami” comprises a long single-strand DNA oligomer connected to a large number of other DNA staple strands.¹ The one-pot synthesis method can accurately provide in high yield many different static DNA frameworks of specific sizes (50–500 nm) and stiffnesses. Furthermore, many different functional groups can be attached to the staple strands, which are chemically synthesized, such as small molecules (like drug substances, fluorescent dyes, and chemical-stimulus responsive chemicals), nanoparticles, and enzymes. All these features combined enable nanoscale control of molecular positioning and dynamic motion.¹

Researchers have produced large DNA frameworks using a number of stepwise approaches, with a common approach leveraging sticky-end hybridization, or the addition of ssDNA (including tiles) to each origami edge.¹ A second method is blunt-end stacking, in which uses the complementarity of DNA to combine different smaller structures together into larger ones. The ability to incorporate chemicals that respond to different stimuli into these larger static structures is particularly noteworthy, as it can allow “sophisticated nanomechanical motion.”

Indeed, several advances have been made with respect to dynamic DNA-based molecular machines. A common approach is to integrate s toehold-mediated DNA strand displacement (TMSD) element (usually an ssDNA linked to a partly dsDNA in its unhybridized domain in which the complementary ssDNA and dsDNA are exchanged) into static DNA nanostructures to form complex architectures, such as nanoboxes, nanotweezers, and nanotubes, that can exhibit real-time coordinated motion and enable programmable DNA-based molecular machines with features that mimic those found in macroscopic machines.

Another common approach to creating dynamic DNA-based molecular machines is by using enzymatic reactions (using nuclease, DNAzyme, and polymerases, for example) to reconfigure architectural components. Many DNA walkers, which have potential applications in next-generation sensors, drug-delivery platforms, and biological computing, have been constructed using this technology. Functional nucleic acids, such as DNA triplex, aptamer, G-quadruplex, or i-motifs, are used to enhance the complexity of dynamic DNA-based molecular machines. They can respond to a variety of different stimuli types and thus have potential use in diagnostics and other sensor applications.

Several different substrates have been used to product surface-bound DNA-based molecular machines, including gold nanoparticles, DNA frameworks, micelles, vesicles, and lipid bilayer membranes. The latter have potential applications in biomedical research and drug delivery, as attachment of DNA-based molecular machines to lipid bilayers allows simulation of intracellular communication and transport in cells. Some researchers have successfully enabled targeted, small molecule drug delivery and influenced cell–cell adhesion behavior and thus recognition of specific types of cells.

Many Potential Applications of Synthetic Molecular Machines in Medicine

One type of synthetic molecular machine receiving particular interest in the biopharma industry is the nanovalve, which has the potential to enable smart drug delivery.⁷ Nanoimpellers are another. Nanovalves comprise a stalk covalently bound to pore openings and a macrocyclic capping agent, such as a cyclodextrin. The pores contain small molecule drug substances and are blocked by the capping agent, which is moved by some form of mechanical motion in response to a specific stimulus, leading to controlled release of the drug substance. Nanovalves are being explored in conjunction with mesoporous silica for drug delivery, in some cases in conjunction with nanoimpellers based on azobenzene tethers to drive the drug substances out of the pores.

Suggested applications for DNA-based molecular machines include biomolecular rulers; high-resolution imaging methods for gathering data at the single-molecule level, such as on DNA movement during genome-processing reactions, molecular mechanisms of protein-mediated reactions, and binding behavior of immunoglobulin Gs; biomolecular factories for the synthesis of bioactive molecules; the transport of molecules to enable decision making and information processing; biosensing for use in medical and other diagnostics; and, as previously mentioned, smart or intelligent drug delivery, as well as programmable medicines.¹

Molecular machines of various types are also under investigation as agents for tissue regeneration and repair through the delivery of growth factors and/or scaffolds on which new tissue can form.⁸ It has also been proposed that molecular machines could be incorporated into wearable devices to facilitate continuous, real-time monitoring of vital signs and biomarkers and used in rapid diagnostic tests performed on-site, eliminating the need for laboratory testing. Overall, molecular machines have the potential to aid in earlier disease detection, reduce treatment side effects, accelerate healing, and in general improve the overall patient experience.

Selected Examples of Therapeutic Molecular Machines

Developing molecular machines for therapeutic applications, most notably targeted and controlled drug delivery, requires the design of machines that can perform as desired within the highly complex human body.⁷ Such molecular machines must be designed, constructed, and tested, all of which requires collaboration among engineers, biologists, medicinal chemists, physicists, and so on.

In some cases, researchers are leveraging cellular stimuli to set molecular machines in motion and enable targeted delivery with enzymatic control.⁷ Nanovalves activated with a change in pH are also being investigated. A more recent example is a combination cancer therapy in which engineered DNAzyme molecular machines with an aptamer and i-motif promote cancer apoptosis via dynamic inter- and intracellular regulation. The aptamer recognizes the cancer cells, and the i-motif shortens the intercellular distance, allowing release of T cells trapped in the tumor microenvironment via metal ion–activated DNAzyme cleavage.⁹ In a similar vein, the same research group created a DNAzyme-based molecular machine with mitochondria-targeted peptides that, when delivered into cancer cells causes mitochondria aggregation, which enables enhanced killing in zinc-deficient cancer cells.

Molecular machines have also been developed as antibiotic therapies that completely avoid the problem of antibiotic resistance.¹⁰ Several different visible light–activated synthetic molecular machines were designed to kill Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, in minutes by physically disrupting the bacterial membranes. They were also shown to enhance the activity of standard antibiotics when used at lower doses.

Rapidly Advancing Field

In just the past few years, many new types of synthetic molecular machines have been designed and constructed using a variety of materials, including liquid crystals, various photosensitive organic molecules, DNA, and others. Some of these artificial molecular machines have highly complex structures and functionalities. Particularly interesting are molecular machines that are attached to surfaces and exhibit directionality in motion and respond “intelligently” to various stimuli.

In the pharmaceutical industry, one of the most exciting potential applications of synthetic molecular machines is their use for drug delivery. Indeed, novel nanoparticle-based drug-delivery systems leveraging artificial molecular machines that impart controlled release and targeting capabilities have been developed.

This field is still in its nascent stages, however, and much more R&D is needed to improve the capabilities of synthetic molecular machines. For instance, achieving the level of functioning observed in natural biomolecular machines requires the incorporation of numerous features into artificial counterparts, including “alignment, order, directionality, tracks, signaling, communication, compartmentalization, amplification, fuel, regeneration, replication, waste management, temporal and spatial control, and feedback loops” at a minimum.⁶ These attributes are prerequisites for individual molecular machines, which then must integrate with one another and the external environment to generate truly useful artificial molecular machines.

Given the progress made to date in techniques for constructing artificial molecular machines and the increased understanding of natural biomolecular machines, these advances will be realized sooner rather than later, and synthetic molecular machines will find many different uses, including for drug delivery and potentially in new classes of therapeutics.

References

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