Researchers have shown that Escherichia coli RNA polymerase can transcribe an expanded genetic alphabet containing eight nucleotide letters instead of the four found in natural DNA. The study, published in Nature Communications, also identified a way to reduce errors involving one of the synthetic bases.
Natural DNA stores information using four bases: adenine, thymine, cytosine, and guanine. The “hachimoji” system – named after the Japanese words for “eight letters” – adds four synthetic bases called P, Z, B, and S. These form two additional pairs, P:Z and B:S.
Expanded alphabets could eventually support the development of nucleic acids with properties that natural DNA and RNA do not possess. Potential applications include engineered RNA molecules, laboratory reagents, and aptamers designed to bind specific molecular targets. However, the technology remains experimental and has not been established for clinical use.
Researchers investigated whether bacterial RNA polymerase could accurately recognize and copy the synthetic bases into RNA. Previous research had shown that the enzyme could process the B:S pair, but its recognition of P:Z had not been fully characterized. The researchers created DNA and RNA templates containing each synthetic base at a defined position. They then tested whether E. coli RNA polymerase selected the correct matching nucleotide from a panel containing the four natural and four synthetic nucleotides.
The enzyme efficiently incorporated the intended synthetic nucleotide opposite each template base. The two synthetic pairs also remained separate from one another, meaning each base preferentially matched with its designated partner.
The rate of P:Z incorporation was approximately twofold lower than that of a natural guanine-cytosine pair under the same experimental conditions. This indicated that the bacterial enzyme could process the synthetic pair at a rate approaching that of natural nucleotides.
One problem involved Z, which sometimes paired incorrectly with guanine. The researchers linked this error to the chemical structure of Z, which can change under conditions close to physiologic pH and adopt a form capable of binding guanine.
To reduce this mismatch, the team tested a modified base called Z*. A nitro group in Z was replaced with a carboxamide group, making the unwanted chemical change less likely. Transcription assays showed that Z* substantially reduced incorrect pairing with guanine while retaining its ability to pair with P.
The researchers noted that their single-nucleotide experiments may overestimate error rates. Each nucleotide was tested separately, without competition from the correct matching nucleotide. In a complete transcription reaction, the intended nucleotide would be expected to compete with and potentially outpace the incorrect one.
Using cryo-electron microscopy, the team also produced four structures of RNA polymerase interacting with the synthetic bases at resolutions of 2.42 to 2.75 angstroms. The structures showed that P:Z and P:Z* fit into the enzyme’s active site in a similar way to natural base pairs.
The synthetic pairs also triggered movement of the polymerase’s “trigger loop,” a structural component that helps the enzyme select and incorporate nucleotides. Additional analysis showed how small chemical differences between Z and Z* affected the enzyme’s conformation and activity.
The study was conducted in a cell-free system. It did not show that a complete eight-letter gene could be transcribed inside living bacteria or translated into a protein. Further work will be needed to improve accuracy and determine whether the expanded alphabet can function reliably in cells.
This research provides a clearer understanding of how existing biological machinery handles synthetic genetic material. It also demonstrates how biochemical testing and cryo-electron microscopy can identify transcription errors and guide the design of more accurate synthetic nucleotides.
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