An Escherichia coli enzyme transcribed eight-letter DNA into RNA in vitro

An Escherichia coli enzyme transcribed eight-letter DNA into RNA in vitro

In an article published on September 2, purified Escherichia coli RNA polymerase, the enzyme that copies DNA into RNA, read synthetic DNA containing four natural and four artificial letters in vitro. It incorporated the letters P, Z, B, and S into RNA and continued extending the RNA after a P:Z pair.

Natural DNA encodes genetic information with four letters: A, T, G, and C. In 2019, researchers created the Hachimoji system by adding P, Z, B, and S. The new letters form P:Z and B:S pairs while preserving the shape of the double helix. At that time, an engineered T7 viral polymerase transcribed the complete eight-letter set into RNA.

The next question concerned a different type of enzyme. In cells, multisubunit RNA polymerases transcribe DNA into RNA. Dong Wang's team assembled Escherichia coli RNA polymerase with short fragments of synthetic DNA and tested all eight nucleotides used to build the RNA. The enzyme incorporated the correct P:Z and B:S pairs particularly efficiently. Under the same conditions, P:Z incorporation was about half as fast as G:C incorporation, after which the enzyme continued synthesizing a long RNA molecule.

The researchers did, however, identify a specific error involving Z. Under some conditions, Z loses a proton, a positively charged particle, and can pair with G instead of P. In the Z* variant, the nitro group of Z was replaced with a carboxamide group. Incorrect G incorporation fell sharply, while correct pairing with P was preserved.

Four cryo-electron microscopy images, obtained using a method that reveals the structures of frozen molecular complexes, provided a view of the enzyme region where the next letter is incorporated. The P:Z and P:Z* pairs retained the geometry typical of DNA at this site. In the Z* series, the researchers observed both the open and closed conformations of the polymerase trigger loop. Closing this loop moves the enzyme into a catalytically active conformation. In the complex containing P:Z, a water molecule connected the nitro group of Z to a bend in the protein backbone within the active site. This interaction helped stabilize a conformation that facilitates trigger-loop closure.

The transition from an engineered viral polymerase to a cellular-type enzyme provides another test of the eight-letter alphabet during the transfer of information from DNA to RNA. This alphabet gives RNA additional chemical capabilities that can be used to create and select aptamers, short molecules that recognize specified substances.