Objective:

To investigate the ability of Escherichia coli RNA polymerase to transcribe an expanded genetic alphabet containing eight nucleotide letters.

Approach:

• Study Design: The researchers created DNA and RNA templates with synthetic bases and tested the incorporation of these bases by E. coli RNA polymerase.
• Error Reduction: A modified base called Z* was tested to reduce incorrect pairing with guanine.
• Structural Analysis: Cryo-electron microscopy was used to produce structures of RNA polymerase interacting with synthetic bases.

Key Findings:

• E. coli RNA polymerase can efficiently incorporate synthetic nucleotides P, Z, B, and S.
• The incorporation rate of the synthetic pair P:Z is approximately twofold lower than that of natural guanine-cytosine.
• The modified base Z* significantly reduces incorrect pairing with guanine while maintaining pairing with P.
• Cryo-electron microscopy revealed that synthetic pairs fit into the enzyme's active site similarly to natural base pairs.

Interpretation:

The study enhances understanding of how biological machinery interacts with synthetic genetic material.

Limitations:

• The study was conducted in a cell-free system and did not demonstrate transcription in living bacteria.
• Single-nucleotide experiments may overestimate error rates due to lack of competition from correct matching nucleotides.

Conclusion:

Further research is needed to assess the functionality of the expanded genetic alphabet in living cells.

Sources:

• Nature Communications