Invention of DNA "Page Numbers" Opens Up Vast Possibilities for the Bioeconomy

The power of artificial intelligence (AI) and advanced computing has made it possible to design genetic sequences encoding for diverse biological applications, such as proteins that form the building blocks of materials stronger than steel, or personalized cancer treatments. But the act of constructing DNA sequences to realize those designs has been a significant bottleneck. Due to technological limitations, chemical DNA synthesis has been limited only to creating short pieces of DNA. However, DNA molecules on the scale of genes or genomes can be tens to thousands of times longer than current capabilities allow. Without DNA construction, AI-powered biological designs cannot be verified or improved—meaning that the blueprints for futuristic new technologies cannot be realized.

Caltech researchers have invented a new technology to write long sequences of DNA with groundbreaking accuracy. The invention, a method called Sidewinder, utilizes the conceptual equivalent of page numbers for DNA, enabling researchers to stitch together any arbitrary number of short pieces of DNA, called synthetic oligonucleotides, or "oligos," in the correct order to create a much larger piece of DNA—up to the scale of a gene or potentially an entire genome (the entire genetic complement of an organism). Synthetic oligos, which are cheap and widely available, can now be combined into any design using Sidewinder. This innovation clears a major bottleneck for bioengineering new compounds and materials, and it could have a vast array of applications, including agriculture and therapeutics.

The invention and development of Sidewinder was conducted in the laboratory of Kaihang Wang, an assistant professor of biology and biological engineering. A paper describing the technique appears in the journal Nature on January 21.

"DNA is the source code of all earthly life and biological functions," Wang says. "As such, biomedical applications and the future bioeconomy depend on the ability to write DNA. Sidewinder provides a new path to the ancient and persisting desire of humankind to rewrite the very source code of life. We can now make long DNA, regardless of complexity and sequence, and do this faster, more easily, and more cheaply than has been possible."

The diversity of life forms on Earth comes from evolution: As organisms replicate, their DNA is copied into their offspring. Over millions of years, mutations accumulate, giving rise to new organisms with new functions. However, this means that in nature, entirely new DNA sequences are never written from scratch—they are only iteratively copied and edited from a pre-existing template.

Humans have taken advantage of the evolutionary process for millennia; for example, over the course of about 9,000 years, humanity domesticated and selectively bred maize. With the advent of modern biology, researchers began to explore the possibility of synthesizing new DNA sequences from scratch for the first time in human history.

In the 1970s, researchers developed the ability to synthesize short pieces of DNA—the aforementioned oligos. These pieces are around 10 to 100 base pairs long. However, attempts to accurately synthesize oligos longer than a few hundred nucleotides were not successful. Genes that encode for many useful proteins are on the order of thousands to tens of thousands of nucleotides, far too long for current synthesizing methods.

In the past four decades, short synthetic oligos have been produced at scale to enable biotech advances, such as the recent messenger RNA (mRNA) vaccines targeting COVID-19. But in order to create entire genes, and thus enable biotechnologies such as personalized cancer vaccines, these short oligos would need to be stitched together with perfect accuracy into longer, complex DNA sequences.

Enter Kaihang Wang's Sidewinder. With Sidewinder, synthetic biologists can write new genes and entire genomes within just a few days, if not hours.

To explain Sidewinder's mechanism, Kaihang uses the analogy of assembling printed pages into books in the West. "In 1441, Johannes Gutenberg invented the printing press, enabling the creation of individual printed pages," he says. "But Gutenberg's bibles never contained any page numbers. These books were painstakingly assembled by aligning the contexts at the beginning and end of each page, for hundreds of pages. Thus, the eventual invention of page numbers, which took about 50 years, was revolutionary. The seemingly simple concept of page numbers was actually non-trivial. We are fortunate to learn from this history, because we are facing a surprisingly identical challenge: how to guide the construction of long sequences from individually synthesized pieces. In this case, they are DNA oligos instead of printed pages. It has been 40 years since the invention of oligo synthesis, the equivalent of a Gutenberg press for DNA, and now it is high time that we invent the equivalent of 'DNA page numbers' to guide the assembly of these short oligos to construct long and functional DNA—the equivalent of books—for genes, gene clusters, and all the way to genomes to complete the book of life."

The Sidewinder process attaches DNA "page numbers" to each oligo, enabling each piece to match up with the right neighbors in the sequence—piece number 4, for example, follows piece number 3 and precedes piece number 5. It works by using a technique called a 3 Way Junction (3WJ), which causes the page number pieces to stick out of the side of the assembled DNA construct like little tags. In this way, the information guiding the construction of the DNA sequence can be separated from the information encoded in the DNA sequence. These little tags extending from the 3WJ are then smoothly removed, resulting in a perfectly assembled, uninterrupted DNA double helix for any desired applications. The ability to remove the third helices from the 3WJs in a single, seamless step after using them as "page numbers" to guide the DNA construction is a critical part of the Sidewinder invention. Guided by the removable DNA page numbers, Sidewinder achieves an incredibly high fidelity in DNA construction with a measured misconnection rate of just one in one million, a four to five magnitude improvement over all prior techniques whose misconnection rates range from 1-in-10 to 1-in-30.

"Sidewinder is wonderfully creative, and a powerful step toward the goal of writing DNA of any complexity," says Caltech's Frances Arnold, the Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry, director of the Donna and Benjamin M. Rosen Bioengineering Center, and winner of the 2018 Nobel Prize in Chemistry. Arnold invented a method called directed evolution—a technology to mimic natural selection in the laboratory—and is now using AI to facilitate protein design. "Sidewinder addresses a key bottleneck in translating computational design into reality, with applications across health and sustainability."

"A cell is a complex computer, one we want to be able to engineer for our own applications," says the study's first author, Noah Robinson (PhD '25), who completed his graduate studies in Kaihang Wang's laboratory and is now continuing the work as a postdoctoral scholar. "To do that, we need to be able to design and construct in the language of life, DNA. With Sidewinder, our lab is developing tools to make this a reality."

The team now plans to address additional bottlenecks in scaling up construction of sequences. Additionally, Kaihang emphasizes the importance of interfacing Sidewinder with AI.

"Interfacing with AI will give us the freedom both to design and to construct," Kaihang says. "Whatever can be designed by AI as a biological function can be constructed through Sidewinder. The convergence of design through AI and construction through next-generation DNA writing technologies such as Sidewinder may enable an alternative to how life can come around, in addition to evolution. Now maybe it can be designed and made."

A paper describing the research is titled "Construction of complex and diverse DNA sequences using DNA 3-Way Junctions." In addition to Robinson and Wang, Caltech co-authors are graduate students Weilin Zhang, Bryan Gerber, Hanqiao Zhang, and Sixiang Wang; and research scientist Charles Sanfiorenzo (PhD '24). Additional co-authors are Rajesh Ghosh and Dino Di Carlo of UCLA. Funding was provided by the National Science Foundation, the Shurl and Kay Curci Foundation, the National Institutes of Health, and Caltech's Center for Environmental Microbial Interactions. Genyro, a biotechnology company co-founded by Kaihang Wang, has entered into an exclusive licensing agreement for Sidewinder to advance next-generation DNA construction.