Revamped design could transport powerful biological computers from test tube to cell

Tiny biological computers made of DNA could revolutionize the way we diagnose and treat a host of diseases, once the technology is fully fleshed out. However, a major stumbling block for these DNA-based devices, which can work in both cells and liquid solutions, is their short lifespan. One use and the computers are exhausted.

Now, researchers at the National Institute of Standards and Technology (NIST) may have developed long-lived biological computers that could potentially persist inside cells. In a paper published in the journal Science Advances, the authors forgo the traditional DNA-based approach, opting instead to use nucleic acid RNA to build computers. The results demonstrate that RNA circuits are as reliable and versatile as their DNA-based counterparts. Additionally, living cells may be able to create these RNA circuits continuously, which is not easily possible with DNA circuits, further positioning RNA as a promising candidate for powerful and durable biological computers. .

Much like the computer or smart device you’re probably reading this about, biological computers can be programmed to perform different types of tasks.

“The difference is that instead of coding with ones and zeros, you write strings of A, T, C and G, which are the four chemical bases that make up DNA,” said Samuel Schaffter, postdoctoral researcher at NIST and lead author of the study. .

By assembling a specific sequence of bases into a strand of nucleic acid, researchers can dictate what it binds to. One strand could be engineered to attach to specific pieces of DNA, RNA, or certain disease-associated proteins and then trigger chemical reactions with other strands in the same circuit to process the chemical information and eventually produce some sort of useful output.

This output could be a detectable signal that could help medical diagnostics, or it could be a therapeutic drug to treat a disease.

However, DNA is not the strongest material and can quickly fall apart under certain conditions. Cells can be hostile environments, as they often contain proteins that chop nucleic acids. And even if the DNA sequences stick around long enough to detect their target, the chemical bonds they form render them useless afterwards.

“They can’t do things like constantly monitor gene expression patterns. They’re single-use, which means they only give you a snapshot,” Schaffter said.

Also being a nucleic acid, RNA shares many problems with DNA when it comes to being a building block of the biological computer. It is susceptible to rapid degradation, and after one strand chemically binds to a target molecule, that strand is terminated. But unlike DNA, RNA could be a renewable resource under the right conditions. To take advantage of this advantage, Schaffter and his colleagues first had to show that RNA circuits, which cells would theoretically be able to produce, could work as well as DNA-based ones.

The advantage of RNA over DNA stems from a natural cellular process called transcription, in which proteins continuously produce RNA using a cell’s DNA as a template. If the DNA in a cell’s genome coded for the circuitry components of a biological computer, the cell would continually produce the computer components.

In the process of biological computation, single strands of nucleic acids in a circuit can easily end up bound to other strands in the same circuit, an undesirable effect that prevents circuit components from binding to their intended targets. The design of these circuits often means that different components will naturally fit together.

To avoid unwanted binding, DNA sequences that are part of computers known as strand displacement circuits are usually synthesized (in machines rather than cells) separately and in double-stranded form. With each chemical base in each strand bound to a base in the other, this double strand acts like a locked door that would only unlock if the target sequence arrived and took the place of one of the strands.

Schaffter and Elizabeth Strychalski, head of NIST’s cell engineering group and co-author of the study, sought to mimic this “locked door” function in their RNA circuitry, keeping in mind that by ultimately the cells would have to produce these locked doors themselves. To prepare the cells for success, the researchers wrote the sequences so that one half of the strands could bind to the other half. By binding in this way, the RNA sequences would fold in on themselves like a hot dog bun, ensuring that they are in a locked state.

But to work properly, the gates would have to be two chemically bonded but separate strands, more like a hamburger bun or a sandwich than a hot dog bun. The team achieved the double-stranded design in their gates by coding in a stretch of RNA called the ribozyme near the gates’ folding point. This particular ribozyme – taken from the genome of a hepatitis virus – is said to separate after the strand of RNA it was embedded in bends, creating two separate strands.

The authors tested whether their circuits could perform basic logical operations, such as unlocking their doors only in specific scenarios, such as if one of two specific RNA sequences were present or only if both were present at the same time. They also built and examined circuits consisting of multiple gates that performed different logical operations in series. Only when these circuits met the right combination of sequences did their doors unlock one by one like dominoes.

The experiments involved exposing different circuits to bits of RNA – some of which the circuits were designed to attach to – and measuring the output of the circuits. In this case, the output at the end of each circuit was a fluorescent reporter molecule that would light up once the final gate was unlocked.

The researchers also tracked how quickly the doors unlocked as the circuits processed inputs and compared their measurements to computer model predictions.

“For me, these had to work in a test tube as predictively as DNA computing. The good thing with DNA circuits is that most of the time you can just write a sequence on a piece of paper, and it will work like that.” you want,” Schaffter said. “The bottom line here is that we found RNA circuits to be very predictable and programmable, much more so than I thought, actually.”

The similarities in performance between DNA and RNA circuits might indicate that it may be advantageous to switch to the latter, as RNA can be transcribed to reassemble the components of a circuit. And many of the existing DNA circuits that researchers have already developed to perform various tasks could theoretically be replaced by RNA versions and behave in the same way. To be sure, however, the study authors need to push the technology further.

In this study, the authors demonstrated that the transcribable circuits work, but they have not yet produced them using the actual cellular transcription machinery. Instead, the machines synthesized nucleic acids through a process similar to that used to produce DNA for research. To take the next step would require inserting DNA into an organism’s genome, where it would serve as a template for the components of the RNA circuit.

“We then want to put them into bacteria. We want to know: can we integrate circuit designs into genetic material using our strategy? Can we get the same kind of performance and behavior when the circuits are in inside the cells? says Schaffter. “We have the potential.”