Ribonucleosides for an Artificially Expanded Genetic Information System

Abstract

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2′-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson–Crick complement, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this “second-generation” AEGIS Z:P pair to add amino acids encoded by mRNA.

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair

“…[7] More recently, we showed that oligonucleotides built from six-letter AEGIS genetic alphabets can evolve in aD NA matrix under laboratory-created selective pressure to give molecules that bind to breast cancer [14] and liver cancer cells; [15] this success suggested af ull search of the expanded "sequence space" created by the additional genetic "letters". [20] ForR NA,s tructural studies are (if anything) more important than with DNA. [16] In contrast with DNA, RNAhas seldom been examined as amatrix for an expanded genetic alphabet.…”

“…Recent results showed that T7 RNAp olymerase can generate RNA molecules containing Z and P AEGIS ribonucleosides from encoding GACTZP DNAm olecules,a nd reverse transcriptase can synthesize Z and P AEGIS DNAf rom complementary GACUZP AEGIS RNA. [20] ForR NA,s tructural studies are (if anything) more important than with DNA. Because of its extra 2'-hydroxy group,R NA has many more conformational opportunities than DNA.…”

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair

“…Only recently was this work extended to the enzymology of the pair between 6‐amino‐5‐nitro‐3‐(1′‐β‐ D ‐ribofuranosyl)‐2(1 H )‐pyridone and 2‐amino‐8‐(1′‐β‐ D ‐ribo‐furanosyl)‐imidazo‐[1,2‐a]‐1,3,5‐triazin‐4‐(8 H )‐one, called Z and P (Figure 1, right box). Recent results showed that T7 RNA polymerase can generate RNA molecules containing Z and P AEGIS ribonucleosides from encoding GACT ZP DNA molecules, and reverse transcriptase can synthesize Z and P AEGIS DNA from complementary GACU ZP AEGIS RNA 20…”

Towards applications of synthetic genetic polymers in diagnosis and therapy

“…Benner and coworkers, as part of their long-term goal of developing a high-fidelity expanded genetic alphabet as described above, recently evolved a Taq polymerase variant capable of efficiently amplifying sequences containing the artificial Z:P base pair, which relies on an unnatural hydrogenbonding pattern [39]. In addition, they have demonstrated that Z and P can be incorporated into RNA transcripts, a key milestone in the step towards their potential use for genetic code expansion [40,41 ]. Recently, the Brakmann laboratory realised a long sought goal of engineering a T7 polymerase capable of synthesising RNA modified with all four 2 0 -O-methyl nucleotides [42].…”