Ribonucleosides for an artificially expanded genetic information system.

Area of Science:

• Synthetic biology
• Nucleic acid chemistry
• Biochemistry

Background:

• Artificially expanded genetic information systems (AEGIS) aim to increase the diversity of genetic polymers.
• Standard DNA and RNA use four nucleobases (A, T, C, G or A, U, C, G).
• Orthogonal base pairs are crucial for AEGIS expansion.

Purpose of the Study:

• To report the large-scale synthesis of a novel AEGIS nucleotide, 2-amino-3-nitropyridin-6-one (Z).
• To evaluate the stability of the ribose form of Z (RiboZ) compared to its deoxyribose analogue.
• To demonstrate the enzymatic incorporation of the Z:P base pair into RNA.

Main Methods:

• Heck coupling and hydroboration/oxidation sequence for Z nucleotide synthesis.
• Epimerization stability assays for RiboZ and its deoxyribose analogue.
• T7 RNA polymerase-mediated incorporation of ZTP opposite its complement P (imidazo[1,2-a]-1,3,5-triazin-4(8H)one).

Main Results:

• Successful large-scale synthesis of the AEGIS nucleotide Z.
• RiboZ demonstrated enhanced stability against epimerization compared to its 2'-deoxyribo counterpart.
• T7 RNA polymerase efficiently incorporated ZTP opposite its Watson-Crick complement P, forming a Z:P base pair in RNA.

Conclusions:

• The Z:P base pair represents a promising second-generation AEGIS component.
• The stability and enzymatic incorporation of Z:P lay the groundwork for expanding the genetic code.
• This advancement facilitates the potential to encode additional amino acids using mRNA.