Artificially expanded genetic alphabet evolves enzymes for the first time

Artificially expanded genetic alphabet evolves enzymes for the first time

An artificially extended DNA alphabet developed enzymes for the first time. The work, which added two synthetic building blocks to DNA’s usual four to create a library of molecules, demonstrates how these libraries could offer improved chemical diversity over standard DNA for finding relevant biomedical molecules, as well only clues to how life began.

As DNA typically has only four bases – A, C, T and G – the richness of possible sequences for building molecular libraries is limited, especially with shorter oligonucleotide sequences, or oligos. One solution is to add functional groups to natural nucleotides to find new molecules. But an alternative approach that has emerged over the past two decades is to extend the genetic alphabet beyond four bases using synthetic nucleotides.

In recent years, researchers have shown that synthetic bases can extend the genetic alphabet, potentially up to 12 bases. However, expanded genetic alphabets had, until now, only produced functionally limited oligos or aptamers that bind to a desired target and were not catalytic.

Now, a team led by Steven Benner and Elisa Biondi of the Foundation for Applied Molecular Evolution in Florida has compared the performance of an artificially extended genetic information system (Aegis) – pioneered by Benner 20 years ago – against standard DNA to the evolution of a reservoir of ribonucleases, enzymes that degrade RNA. During this process, Aegis developed a new type of ribonuclease, dubbed “Aegiszyme”.

“We hadn’t yet demonstrated beyond doubt that expanded DNA performs better than standard DNA, because we hadn’t done a parallel experiment before,” says Biondi. “We now have evidence that our expanded DNA approach rests on a solid foundation. The result [of a new catalyst] was hoped for if not entirely surprising, nor expected.


Aegis included the standard DNA bases plus two additional bases, Z and P. Separate 25 nucleotide long libraries were then constructed for Aegis and ordinary DNA. The libraries were then subjected to identical and repeated cycles of selective pressure with respect to ribonuclease activity.

The team found that even after 16 rounds of selection exploring 25% of possible sequence combinations, the standard DNA library showed no ribonuclease activity. Conversely, the Aegis experiment investigated only 0.0011% of the possible sequence space and revealed enhanced RNA cleavage activity.

“While it is unclear whether these early Aegiszymes outperform the current generation of DNAzymes and XNAzymes, it is certainly of interest that they can be selected from relatively short oligo libraries,” comments synthetic biologist Alex Taylor of Cambridge University, UK. “For potential therapy, a shorter catalytic oligo would be beneficial to enable efficient chemical synthesis and could offer, for example, tissue penetration or enhanced cellular uptake.”

“The work is of obvious practical value because it shows how an expanded genetic alphabet can help discover aptamers with rare properties,” says Floyd Romesberg, whose Scripps Research lab successfully inserted Unnatural DNA in a bacterium in 2017. “This should open up the possibility of discovering other aptamers with unique properties that have proven difficult to discover.

The fact that short sequences of an extended alphabet can evolve catalysts also offers insight into the origin of life. “In prebiotic times, when nucleic acids or protonucleic acids were thought to serve both genetic and catalytic functions, early evolving Darwinian systems may have used a wider variety of chemical groups than we see now in the molecules of life. ‘, says Biondi. “In doing so, they could have gotten away with shorter polymer lengths, an advantage both in terms of energy expenditure and resource availability.”

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