DNA machine, go ahead

2016 The Bell Prize in Chemistry was awarded to three chemists who studied molecular machines. The reason for the award was "the design and synthesis of molecular machines." They created molecular machines through organic molecule synthesis, bringing science fiction into reality. Now using programmable biological material - DNA, biochemists and biophysicists create DNA machines with various functions. Let’s take a look at what they’ve created in these cutting-edge laboratories, DNA robots? Will they be satisfied with this?

Author | Nina Notman (science writer)

Translation | Ji Province

Nucleic acid The machine is getting better.

——Nina Notman

There are millions of molecular motors and various "machines" in the human body— —These machines maintain the breathing of our lungs, the beating of the heart, the thinking of the brain, and the peristalsis of the digestive system... "Life is so magical. At the microscopic level of life, there are all kinds of incredible functions. We understand The more you use it, the more you want to reproduce these functions." William Shi, professor of biochemistry at Harvard University( William Shih) said.

The enlightenment work often mentioned by researchers in this field was released in 1966 The science fiction movie "Magic Journey"(Fantastic Voyage)< /span>. There is a submarine in the movie. After it and the entire crew are shrunk, they are sent into the body of an injured scientist to repair his brain. This movie gives people hope that one day in the future, artificial molecular machines can easily enter the body to complete tasks such as drug delivery, disease diagnosis, and surgical operations. Molecular machines also have uses outside of medicine, such as as analytical tools or nanofactories for compound synthesis.

Today, there are two types of materials used to build molecular machines: one is synthetic Organic molecules, the pioneers in this field won the Nobel Prize in Chemistry in 2016; the second is biological materials, which is the core content of this topic.

Like children's building blocks

Nature mainly uses proteins as parts to build molecular machines, while researchers use DNA. Shi explained, "It's easier to program DNA to create different shapes and functions than proteins."

DNA stores genetic instructions in a sequence composed of four bases. Medium adenine（adenine,A）, thymine(thymine, T), guanine(guanine, G), cytosine(cytosine, C)along Skeleton arrangement. Single strands of DNA always assemble themselves into double strands in a predictable way: A's pair up with T's, G's pair up with C's. Friedrich Simmel, Professor of Biophysics at the Technical University of Munich, Germany（Friedrich Simmel）< span style="color: #3F3F3F; --tt-darkmode-color: #A3A3A3;"> explained that when DNA is used as a "building material", researchers can treat the base sequence on the DNA chain as a Code, code stipulates that one part of the DNA strand must adhere to another part. "We can basically ignore the complexity of chemistry because everything is done automatically and reduced to one line of code."

The first person to think of using DNA as a "building material" was Nana from New York University in the United States. Ned Seeman（Ned Seeman）, that was in the early 1980s. Since then, a range of techniques have been developed for building every conceivable DNA structure. These techniques include DNA origami(DNA origami), this is Paul Rosemond of California Institute of Technology(Paul Rothemund)'s idea: using short strands of DNA as "staples" , folding long single-stranded DNA into complex three-dimensional shapes. His team also produced DNA tiles(DNA tiles)and bricks(DNA bricks) , these things can be put together like Lego blocks.

Reference illustration: Lego-type DNA structure丨Source: Science

Over the past few years, two of the biggest hurdles for DNA nanotechnology have been size and Stability - got a breakthrough. Hendrik Dietz, Professor of Biophysics at the Technical University of Munich(Hendrik Dietz) said that before this, it was very difficult to design self-assembled DNA structures exceeding 100 nanometers. Dietz and Yin Peng from Caltech in 2017(Translation: Currently working at Harvard University) and Qian Lulu announced three different strategies at the same time, which broke this barrier.

The DNA backbone is negatively charged, which means that when the folded DNA is close to each other , they will instinctively repel each other, causing the structure to spread apart. In the laboratory, repulsive forces can be counteracted by placing DNA in a highly cationic solution. However, the human body does not provide DNA with such an ideal paradise. In the human body, DNA structures are constantly attacked by DNA enzymes, which are designed to degrade DNA molecules. Dietz explained, "What we have to do is find a way to stabilize the DNA folding structure. . This way, when we move from the ideal conditions of experiments to practical applications, these structures can still Keep it." Both Dietz's team and Shi's team have recently developed methods to stabilize the DNA structure. They use chemical cross-linking methods to connect the DNA structures together. "Now it's finally possible to keep these structures in the body for weeks or longer," Shi said.

Have a little "little swing"

A large stable structure alone is not enough to be a machine: a machine is Be able to move. Simon mentioned that a mixture of rigid elements like double-stranded DNA and flexible elements like single-stranded DNA can move around driven by Brownian motion. However, getting them to move in a highly controllable manner is extremely challenging.

The predictable and programmable properties of DNA are important in the self-assembly process, as well The key in the DNA machine. In 2000, Bernie Yurke(Bernie Yurke) And Andrew Turnfield, professor of physics at the University of Oxford, UK(Andrew Turberfield), collaboration reveals first guide Means of DNA movement: DNA-chaperone replacement process, that is, strand displacement reaction. This method is still widely used today. Turberfield explained, "This is to replace all or part of one of the double strands with another strand with the same base sequence."

For example, DNA Walker(walker), you can pass the double The chain repeatedly switches partners as it opens and closes, causing its "feet" to walk up and down along a row of "pegs" with complementary base pairs.

In 2017, researchers at Caltech demonstrated that DNA transport devices can use strands Displacement reactions sort cargo. This DNA "cargo sorter" could wander around a surface, picking up discarded goods and sorting them into different piles; putting colored clothes in one pile and white clothes in the same way we collect dirty laundry at home. .

The surface that the DNA machine walks on is composed of two single strands of DNA. dimensional DNA origami structure. The cargo is single-stranded DNA containing DNA "barcode tags." The robot is a single strand of DNA made up of three parts: "feet" to walk, "arms" to collect and transport goods, and segments to read tags.

The robot will randomly walk on the surface until it hits the cargo. It will pick up the cargo and continue wandering to the unloading point. If the cargo barcode matches the unloading point, the machine will release the cargo from the hands and drop it. "The robot knows where each item is going; Because each shipment will have a DNA barcode, only one of these destinations will match the barcode." Caltech computer scientist Eric ·Winfree（Erik Winfree）explained.

This type of machine was first demonstrated using robots to sort green and red colors Fluorescently stained DNA. However, the research team pointed out that the DNA machine can also sort other small molecules, including aptamers (aptamer, Refers to a type of single-stranded nucleic acid molecules that form a specific three-dimensional spatial configuration through folding), antibodies, small molecules compounds, metal nanoparticles, and proteins.

"Being able to transport objects from point A to point B is very important." Shi explains, "In electronic devices, you can use electric current to change the state of the machine. Similarly, we can use the physical movement of matter to accomplish the same thing."

Speed ​​up the process< /span>

The DNA cargo sorter works very slowly, dividing 6 dye molecules into Two piles will take 24 hours. To speed up the process, the team used multiple robots working simultaneously. Another way is to have the machine go directly from point A to point B instead of doing a random walk. However, this is easier said than done. In order to create a forward biasing force in the system, one way is to use short strands of DNA (also used here called chemical fuel)to participate in the strand displacement reaction. “It’s a tool for designing unbalanced reaction networks,” explains Turberfield, who invented the concept. “It’s a clever kinetic trick that makes the foot behind you lift up more than the foot in front. Faster."

Now, Shi's lab is using chemical fuel to power DNA nanocalipers. These nanocalipers hover around large molecules and measure them. Shi said these clamp-like tools surround different parts of the target, and they cover the target surface at different points by the amount of magnification required, thus marking the length of the target at that location. The ultimate goal is to use these tools to resolve the three-dimensional structure of macromolecules, potentially faster than traditional analytical tools such as X-ray crystallography currently in use. "Instead of directly imaging the molecule, we use these small rulers to measure the distances between all pairs of points. Then we input these distance data into the structure prediction program, and the program can reconstruct the overall three-dimensional shape of the molecule. And get into the details,” Shi explained. He is working with Harvard colleague Wesley Wong(Wesley Wong)Collaborate to develop this tool.

Use "force"

Means to speed up the DNA machine also include: changing the pH value of the solution, adding ions, Use light to excite or apply an electric field. Simon's lab uses this last approach to control DNA robotic arms. Simon said that the modified robotic arm moves 100,000 times faster than the traditional DNA robotic arm.

The robotic arm is a bundle of rigid DNA double strands adhered to by single strands of DNA On the three-dimensional DNA origami platform. Single-stranded DNA is equivalent to a flexible joint that allows the robotic arm to rotate relative to the platform. Since DNA is negatively charged, when the system is subjected to an electric field, the robotic arm moves toward the DNA. Simon also introduced a series of docking stations to the DNA origami platform to increase positional control of the robotic arm. The platform and the arm have matching protruding short strands of DNA that pair up when they are close together, locking the arm in place.

Simon Labs demonstrated that this arm can transport organic fluorescent dyes and Gold nanorods. He explained that the cargo transport device could serve as a molecular version of a factory assembly line, with an external operator issuing instructions to guide the rapid construction of molecules.

Grow me bigger

Another potential application for DNA machines is for controlling macroscopically sized robots. "My research group is making products that can integrate traditional soft materials(such as hydrogels)A machine with dynamic DNA elements that drive its movement." Rebecca Schumann(Rebecca Schulman) said, she He is a professor of biomolecular engineering at Johns Hopkins University in the United States. These robots range in size from 10 microns to 10 millimeters.

Schuman's lab's hydrogel uses DNA double-stranded immobilization; Traditional small molecule cross-linking agent. "Our material contains polyacrylamides that physically cross-link by hybridizing DNA," she said. Her team used a strand displacement reaction to expand the hydrogel, inserting large numbers of hairpin-shaped DNA molecules into the DNA double-stranded cross-links. In the linked structure, the structure is extended, resulting in the growth of the hydrogel. "As this material grows, the size changes are very dramatic," Schumann said. The volume is enlarged 100 times."

The team is now taking advantage of this phenomenon by controlling the timing of enlargement of different parts. Achieve control of the movement of macro-sized robots. They placed different types of DNA hydrogel on different parts of the device. The DNA double strands cross-linked by each hydrogel contain different base sequences, and they will only respond to DNA hairpins with matching sequences. Using this method, the team achieved the folding of designated "petals" on hydrogel "flowers" and also created a hydrogel "crab" whose tentacles, pincers and legs can all respond to different DNA hairpin structures. and bending occurs.

Hydrogel crab丨Source : Johns Hopkins University Rebecca Shuman Laboratory Home Page

The ultimate goal of the team is to design a robot that can move autonomously in response to external stimuli . To move toward this goal, the team is designing receptors that can release DNA hairpins, allowing the device to move when stimulated by specific small molecules. Schumann said, "This is a very initial step not only for the robot, but also for the controller."

Possible application of autonomous motion devices as smart capture devices that can detect chemical gradients and move along this. "With the ability to respond to chemical signals from a distance and move, a robot could find and pick up specific types of cells or debris. Such robots could perform biological biopsies or keep surfaces extremely clean," Schumann said.

Indicate time< /span>

Alyssa Frank at UCLA (Elisa Franco)'s laboratory is focusing on the time control of autonomous DNA machines. "I'm interested in understanding how to make DNA nanostructures change shape in response to stimuli, and how to make this shape change happen regularly over a period of time," she explains.

In 2019, her lab demonstrated that cells can assemble and dissociate rhythmically DNA nanotubes. The basic material of nanotubes is DNA tiles, which have multiple single-stranded DNAs. Nanotube assembly begins when DNA single-stranded complementary strands from different tiles pair up to form double strands; when "invading strands" arrive and disrupt the original pairing, the nanotubes dissociate. Afterwards, the combined invasion strand is replaced by the "anti-invasion strand", which triggers the reassembly of the DNA nanotubes, allowing the DNA tiles to freely form nanotubes again. "We now have nanometer-sized monomers that can grow into micrometer-sized tubular structures in a matter of minutes," Frank said.

The researchers were inspired by the cytoskeleton, which is the cyclic structure during cell division. Sexually growing tubular network. "We want the same actions to happen over and over again," Franck said. Intrusion and anti-invasion chains can be artificially added to the system periodically and repeatedly to control assembly and dissociation. However, to achieve autonomous control, the team combined the system with a synthetic molecular oscillator, which they used to regulate the release of the two DNA strands.

The oscillator consists of a negative feedback genetic loop and two enzymes. The loop is formed by two synthetic DNA genes encoding the invasion strand and the anti-invasion strand; two enzymes are RNA polymerase responsible for making the invasion strand and the anti-invasion strand, and the other is a nuclease that degrades both strands. The oscillatory behavior of genes is driven by the periodic production and degradation of invasion and anti-invasion strands.

Frank hopes that these nanotubes will eventually be used in functional artificial in cells. "At the moment, we are trying to wrap this system into a water droplet, which is the smallest equivalent structure of a cell," she said. -darkmode-color: #A3A3A3;">We are studying how to build an artificial cytoskeleton composed entirely of DNA."

Enter the factory< /span>

The designers of DNA machines were also inspired by another type of cellular machine—— Ribosome. Ribosomes are molecular protein factories, along the lines of messenger RNA(messenger RNA)chain movement, amino acids(with transfer RNA molecule tag)According to the information encoded in the messenger RNA base sequence, in sequence Assemble together.

The researchers used the same concept to guide the synthesis of polymers. Turberfield said, "We and Rachel O'Reilly of Birmingham(Rachel O'Reilly) Together with the team, we are developing molecular machines for genetically programmed polymer synthesis." In this device It was decided that synthetic DNA strands would play the roles of messenger RNA and transfer RNA, with synthetic monomers replacing amino acids. Turberfield explains, "We were given a series of different parts that are recognized by DNA similar to transfer RNA. Our machine is genetically programmed to recognize adapters(adapter) sequence, put these The parts are moved near where the polymers are growing, forcing them to react in a set order."

This autonomous system already connects a variety of natural and non-natural parts to Together: Using peptide bonds and carbon double bonds to connect the parts, purification is not even required. Turberfield is optimistic that this technology will be used in the pharmaceutical industry to rapidly synthesize large-scale combinatorial libraries to discover drug precursors. The idea is to create a soup of trillions of genetically programmed molecules in a reactor, with DNA tags still attached to them. This soup is then used to screen for drug targets; once a positive reaction is found, the DNA tag can be used to identify the molecule that triggered the reaction.

DNA has successfully demonstrated that it can be used as a programmable engineering machine, however , many researchers in the field expect DNA to eventually become a thing of the past. It will be replaced by new natural and artificial materials. These new materials can also self-assemble, but have stronger and more useful functions, such as electromagnetic properties. Shi said, "DNA was an exemplary material during this period of history , we can use it to test various ideas for packaging various functions of life, but in the end we still have to Patterns are thrown into other materials.”

Dietz agrees, "In physics, we often follow this A philosophy: Take a simple system and learn as much as you can, then gradually explore more complex things."

References

1 Original text: https:// www.chemistryworld.com/features/dna-machines-get-a-move-on/4012993.article

2 https://science. sciencemag.org/content/338/6111/1159.full